Effect of Decellularization Protocol on the Mechanical Behavior of Porcine Descending Aorta

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John C. Fitzpatrick

2010-01-01

Full Text Available Enzymatic-detergent decellularization treatments may use a combination of chemical reagents to reduce vascular tissue to sterilized scaffolds, which may be seeded with endothelial cells and implanted with a low risk of rejection. However, these chemicals may alter the mechanical properties of the native tissue and contribute to graft compliance mismatch. Uniaxial tensile data obtained from native and decellularized longitudinal aortic tissue samples was analyzed in terms of engineering stress and fit to a modified form of the Yeoh rubber model. One decellularization protocol used SDS, while the other two used TritonX-100, RNase-A, and DNase-I in combination with EDTA or sodium-deoxycholate. Statistical significance of Yeoh model parameters was determined by paired t-test analysis. The TritonX-100/EDTA and 0.075% SDS treatments resulted in relatively variable mechanical changes and did not effectively lyse VSMCs in aortic tissue. The TritonX-100/sodium-deoxycholate treatment effectively lysed VSMCs and was characterized by less variability in mechanical behavior. The data suggests a TritonX-100/sodium-deoxycholate treatment is a more effective option than TritonX-100/EDTA and SDS treatments for the preparation of aortic xenografts and allografts because it effectively lyses VSMCs and is the least likely treatment, among those considered, to promote a decrease in mechanical compliance.

Quantifying the effects of UV-A/riboflavin crosslinking on the elastic anisotropy and hysteresis of the porcine cornea by noncontact optical coherence elastography

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Singh, Manmohan; Li, Jiasong; Raghunathan, Raksha; Han, Zhaolong; Nair, Achuth; Liu, Chih-Hao; Aglyamov, Salavat R.; Twa, Michael D.; Larin, Kirill V.

2017-02-01

The collagen fibril orientation of the cornea can provide critical information about cornea tissue health because diseases such as keratoconus and therapeutic interventions such as UV-A/riboflavin corneal collagen crosslinking (CXL) can alter the ultrastructural arrangement of collagen fibrils. Here, we quantify the elastic anisotropy and hysteresis of in situ porcine corneas as a function of intraocular pressure (IOP) with noncontact optical coherence elastography. Moreover, the effects of UV-A riboflavin corneal collagen crosslinking on the elastic anisotropy and hysteresis were evaluated. The propagation of an air-pulse induced elastic wave was imaged at stepped meridional angles by a home built phasestabilized swept source OCE system. The stiffness of the cornea was translated from the velocity of the wave, and the elastic anisotropy was quantified by modifying the planar anisotropy coefficient. As the IOP increased, the stiffness of the corneas increased from 18 kPa at 15 mmHg IOP to 120 kPa at 30 mmHg IOP. While there was a measureable hysteresis, it was not significant. After CXL, the Young's modulus of the corneas significantly increased from 18 kPa to 44 kPa at 15 mmHg IOP. The mechanical anisotropy also increased significantly from 10 a.u. in the untreated corneas to 23 a.u. in the CXL treated corneas, 15 mmHg IOP. However, CXL did not change the elastic anisotropic orientation, and the mechanical anisotropic hysteresis was not significant after CXL.

Long-term healing of mildly cross-linked decellularized bovine pericardial aortic patch.

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Umashankar, P R; Sabareeswaran, A; Shenoy, Sachin J

2017-10-01

Glutaraldehyde treated bovine pericardium is extensively used in cardiovascular surgery. However, frequent occurrence of failure modes, such as calcification and structural failure, has hard pressed the need for finding an alternate technology. Decellularized bovine pericardium is an emerging technology. Mildly cross-linked decellularized bovine pericardium promotes positive remodeling with insignificant calcification and acute inflammation. In the present study, mildly cross-linked decellularized bovine pericardium was evaluated as a cardiovascular patch by studying mechanical strength as well as graft remodeling, resistance to calcific degeneration and inflammatory response using long duration porcine aortic implantation. It was observed that decellularized bovine pericardium, although thinner and less elastic had equivalent tensile properties such as tensile strength and stiffness when compared to commercially available glutaraldehyde-treated bovine pericardium. It showed the potential for site appropriate remodeling evidenced by host cell incorporation, thinner neointima, graft degradation, and neocollagenisation making it suitable for vascular patch application, whereas glutaraldehyde-treated pericardium failed to integrate with host tissue through timely degradation and host cell incorporation or neocollagenization. Conversely, it elicited persistent acute inflammation and produced calcification. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2145-2152, 2017. © 2016 Wiley Periodicals, Inc.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

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Balestrini, Jenna L; Gard, Ashley L; Gerhold, Kristin A; Wilcox, Elise C; Liu, Angela; Schwan, Jonas; Le, Andrew V; Baevova, Pavlina; Dimitrievska, Sashka; Zhao, Liping; Sundaram, Sumati; Sun, Huanxing; Rittié, Laure; Dyal, Rachel; Broekelmann, Tom J; Mecham, Robert P; Schwartz, Martin A; Niklason, Laura E; White, Eric S

2016-09-01

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement.

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López-Ruiz, Elena; Venkateswaran, Seshasailam; Perán, Macarena; Jiménez, Gema; Pernagallo, Salvatore; Díaz-Mochón, Juan J; Tura-Ceide, Olga; Arrebola, Francisco; Melchor, Juan; Soto, Juan; Rus, Guillermo; Real, Pedro J; Diaz-Ricart, María; Conde-González, Antonio; Bradley, Mark; Marchal, Juan A

2017-03-24

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

Repair of a common bile duct defect with a decellularized ureteral graft

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Cheng, Yao; Xiong, Xian-Ze; Zhou, Rong-Xing; Deng, Yi-Lei; Jin, Yan-Wen; Lu, Jiong; Li, Fu-Yu; Cheng, Nan-Sheng

2016-01-01

AIM To evaluate the feasibility of repairing a common bile duct defect with a decellularized ureteral graft in a porcine model. METHODS Eighteen pigs were randomly divided into three groups. An approximately 1 cm segment of the common bile duct was excised from all the pigs. The defect was repaired using a 2 cm long decellularized ureteral graft over a T-tube (T-tube group, n = 6) or a silicone stent (stent group, n = 6). Six pigs underwent bile duct reconstruction with a graft alone (stentless group). The surviving animals were euthanized at 3 mo. Specimens of the common bile ducts were obtained for histological analysis. RESULTS The animals in the T-tube and stent groups survived until sacrifice. The blood test results were normal in both groups. The histology results showed a biliary epithelial layer covering the neo-bile duct. In contrast, all the animals in the stentless group died due to biliary peritonitis and cholangitis within two months post-surgery. Neither biliary epithelial cells nor accessory glands were observed at the graft sites in the stentless group. CONCLUSION Repair of a common bile duct defect with a decellularized ureteral graft appears to be feasible. A T-tube or intraluminal stent was necessary to reduce postoperative complications. PMID:28082809

Histological Evaluation of Decellularized Skeletal Muscle Tissue Using Two Different Decellularization Agents

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Hana Hrebíková

2017-02-01

Full Text Available The aim of the present study was to determine effect of two decellularized agents, sodium dodecyl sulphate (SDS and Triton X-100, to the skeletal muscle tissue. Final scaffold was evaluated by several histological techniques to analyse preservation of essential structures including collagen and elastic fibres, basement membranes, glycosaminoglycans and also to confirm elimination of nuclear and cytoplasmic components which are redundant in effectively prepared decellularized scaffolds. Comparison of tissue scaffolds processed with different detergents proved that SDS is superior to Triton X-100 as it can effectively decellularize muscle tissue.

Improvement of the in vivo cellular repopulation of decellularized cardiovascular tissues by a detergent-free, non-proteolytic, actin-disassembling regimen.

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Assmann, Alexander; Struß, Marc; Schiffer, Franziska; Heidelberg, Friederike; Munakata, Hiroshi; Timchenko, Elena V; Timchenko, Pavel E; Kaufmann, Tim; Huynh, Khon; Sugimura, Yukiharu; Leidl, Quentin; Pinto, Antonio; Stoldt, Volker R; Lichtenberg, Artur; Akhyari, Payam

2017-12-01

Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent-free, non-proteolytic, actin-disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio-functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio-functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN-coated DET-decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent-free, non-proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio-functionality. Additionally, this study implies that the actin-disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Decellularized matrices for cardiovascular tissue engineering.

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Moroni, Francesco; Mirabella, Teodelinda

2014-01-01

Cardiovascular disease (CVD) is one of the leading causes of death in the Western world. The replacement of damaged vessels and valves has been practiced since the 1950's. Synthetic grafts, usually made of bio-inert materials, are long-lasting and mechanically relevant, but fail when it comes to "biointegration". Decellularized matrices, instead, can be considered biological grafts capable of stimulating in vivo migration and proliferation of endothelial cells (ECs), recruitment and differentiation of mural cells, finally, culminating in the formation of a biointegrated tissue. Decellularization protocols employ osmotic shock, ionic and non-ionic detergents, proteolitic digestions and DNase/RNase treatments; most of them effectively eliminate the cellular component, but show limitations in preserving the native structure of the extracellular matrix (ECM). In this review, we examine the current state of the art relative to decellularization techniques and biological performance of decellularized heart, valves and big vessels. Furthermore, we focus on the relevance of ECM components, native and resulting from decellularization, in mediating in vivo host response and determining repair and regeneration, as opposed to graft corruption.

Decellularization of placentas: establishing a protocol

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L.C.P.C. Leonel

2017-11-01

Full Text Available Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic.

Click-coated, heparinized, decellularized vascular grafts.

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Dimitrievska, Sashka; Cai, Chao; Weyers, Amanda; Balestrini, Jenna L; Lin, Tylee; Sundaram, Sumati; Hatachi, Go; Spiegel, David A; Kyriakides, Themis R; Miao, Jianjun; Li, Guoyun; Niklason, Laura E; Linhardt, Robert J

2015-02-01

A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through "alkyne-azide" click chemistry, affording a tenfold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. X-ray photoelectron spectroscopy, nuclear magnetic resonance imaging, mass spectrometry and Fourier transform infrared FTIR spectroscopy were used to characterize the synthesis steps, building the final heparin layered coatings. The continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. The efficacy of heparin linkage was demonstrated with factor Xa anti-thrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels. Copyright © 2014 Acta Materialia Inc. All rights reserved.

Terminal sterilization of equine-derived decellularized tendons for clinical use

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Pellegata, Alessandro F.; Bottagisio, Marta; Boschetti, Federica; Ferroni, Marco; Bortolin, Monica; Drago, Lorenzo; Lovati, Arianna B.

2017-01-01

In the last few years, the demand for tissue substitutes has increased and decellularized matrices has been widely proposed in the medical field to restore severe damages thanks to high biocompatibility and biomechanical properties similar to the native tissues. However, biological grafts represent a potential source of contamination and disease transmission; thus, there is the need to achieve acceptable levels of sterility. Several sterilization methods have been investigated with no consensus on the outcomes in terms of minimizing structural damages and preserving functional features of the decellularized matrix for transplantation in humans. With the aim of making decellularized tendons safe for clinical use, we evaluated the cytocompatibility, and biochemical, structural and biomechanical variations of decellularized equine tendons sterilized with peracetic acid or β-irradiation and differently wet- or dry- stored at 4 °C or − 80 °C, respectively. Considering that both sterilization and long-term storage are crucial steps that could not be avoided, our results pointed at ionizing β-rays as terminal sterilization method for decellularized grafts followed by frozen dry storage. Indeed, this approach can maintain the integrity of collagen-based structures and can avoid biomechanical changes, thus making xenogeneic decellularized tendons a promising candidate for clinical use. - Highlights: • A decellularized tendon matrix has been generated. • The sterility of the decellularized matrix is mandatory for transplantation. • β-irradiation and cold storage preserve the matrix structure and biomechanics.

Terminal sterilization of equine-derived decellularized tendons for clinical use

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Pellegata, Alessandro F. [Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milan (Italy); Bottagisio, Marta [Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Via Riccardo Galeazzi 4, 20161 Milan (Italy); Boschetti, Federica; Ferroni, Marco [Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milan (Italy); Bortolin, Monica [Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Via Riccardo Galeazzi 4, 20161 Milan (Italy); Drago, Lorenzo [Laboratory of Clinical Chemistry and Microbiology, IRCCS Galeazzi Orthopaedic Institute, Via Riccardo Galeazzi 4, 20161 Milan (Italy); Department of Biomedical Science for Health, University of Milan, via Luigi Mangiagalli 31, 20133 Milan (Italy); Lovati, Arianna B., E-mail: arianna.lovati@grupposandonato.it [Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Via Riccardo Galeazzi 4, 20161 Milan (Italy)

2017-06-01

In the last few years, the demand for tissue substitutes has increased and decellularized matrices has been widely proposed in the medical field to restore severe damages thanks to high biocompatibility and biomechanical properties similar to the native tissues. However, biological grafts represent a potential source of contamination and disease transmission; thus, there is the need to achieve acceptable levels of sterility. Several sterilization methods have been investigated with no consensus on the outcomes in terms of minimizing structural damages and preserving functional features of the decellularized matrix for transplantation in humans. With the aim of making decellularized tendons safe for clinical use, we evaluated the cytocompatibility, and biochemical, structural and biomechanical variations of decellularized equine tendons sterilized with peracetic acid or β-irradiation and differently wet- or dry- stored at 4 °C or − 80 °C, respectively. Considering that both sterilization and long-term storage are crucial steps that could not be avoided, our results pointed at ionizing β-rays as terminal sterilization method for decellularized grafts followed by frozen dry storage. Indeed, this approach can maintain the integrity of collagen-based structures and can avoid biomechanical changes, thus making xenogeneic decellularized tendons a promising candidate for clinical use. - Highlights: • A decellularized tendon matrix has been generated. • The sterility of the decellularized matrix is mandatory for transplantation. • β-irradiation and cold storage preserve the matrix structure and biomechanics.

Corneal epithelial cell viability of an ex vivo porcine eye model.

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Chan, Ka Yin; Cho, Pauline; Boost, Maureen

2014-07-01

The aim was to assess the consistency of corneal epithelial cell viability of an ex vivo porcine eye model. Six porcine eye models (four test and two control) were prepared for each experiment. The model has a computer-controlled mechanical arm, which could move the eyelid of the porcine eye and apply phosphate buffered saline to simulate blinking and lacrimation. The four test eyes were set up to simulate evaporative dry eyes with simulated lacrimation and blinking (one blink and one drop of buffered saline per minute) over three hours. Control A models were set up to collect pre-experimental baseline data, while those of control B were the same as the test eyes but without lacrimation and blinking simulation. All porcine eyes were kept in a closed chamber with temperature and humidity well controlled. After three hours, the cells of all eyes (except control A, which were assessed immediately before commencement of the experiment) were assessed. The eyes were first dipped into 0.4 per cent trypan blue solution. Following the dissection and separation of the cells, the number of dead cells were then counted under the microscope with a field size of 0.25 mm(2). The experiment was repeated 11 times. No significant differences were found in the number of dead cells among the four test eyes in both the central and peripheral cornea. There were significantly more dead cells in the test eyes compared to control A but significantly less when compared to control B. More dead cells were found in the central cornea than the peripheral cornea in the test eyes but the difference was not observed in controls A and B. Epithelial cell viabilities among the four porcine eye models with simulated lacrimation and blinking were consistent. The majority of cells were viable before the experiment and simulated lacrimation and blinking maintained more viable cells over time. © 2014 The Authors. Clinical and Experimental Optometry © 2014 Optometrists Association Australia.

Comparison of glutaraldehyde and carbodiimides to crosslink tissue engineering scaffolds fabricated by decellularized porcine menisci

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Gao, Shuang; Yuan, Zhiguo; Guo, Weimin; Chen, Mingxue; Liu, Shuyun; Xi, Tingfei; Guo, Quanyi

2017-01-01

The objectives of this study were to fabricate porous scaffolds using decellularized meniscus, and to explore a preferable crosslinking condition to enhance mechanical properties of scaffolds. Moreover, the microstructure, porosity, biodegradation and cytotoxicity were also evaluated. EDAC or GTA in different concentration was used to crosslink scaffolds. FTIR demonstrated functional groups change in crosslinking process. SEM photography showed that crosslinked scaffolds had blurry edges, which resulted scaffolds crosslinked by 1.2 mol/l EDAC had smaller porosity than other groups. The structure change enhanced antidegradation property. After immersing in enzyme solution for 96 h, scaffolds crosslinked by GTA and EDAC could maintain their mass > 70% and 80%. Most importantly, mechanical properties of crosslinked scaffolds were also improved. Uncrosslinked Scaffolds had only 0.49 kPa in compression modulus and 12.81 kPa in tensile modulus. The compression and tensile modulus of scaffolds crosslinked by 1.0% GTA were 1.42 and 567.44 kPa respectively. The same value of scaffolds crosslinked by 1.2 mol/l EDAC were 1.49 and 532.50 kPa. Scaffolds crosslinked by 1.0% and 2.5% GTA were toxic to cells, while EDAC groups showed no cytotoxicity. Chondrocytes could proliferate and infiltrate within scaffolds after seeding. Overall, 1.2 mol/l EDAC was a preferable crosslinking condition. - Highlights: • Porous meniscus scaffolds were fabricated using decellularized meniscus tissue. • Mechanical properties of meniscus scaffolds were enhanced by chemical crosslinking. • The crosslinked scaffold showed enhanced anti-degradation properties. • Chondrocytes could infiltrate and proliferate within crosslinked scaffolds.

Comparison of glutaraldehyde and carbodiimides to crosslink tissue engineering scaffolds fabricated by decellularized porcine menisci

Energy Technology Data Exchange (ETDEWEB)

Gao, Shuang [Center for Biomedical Material and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Yuan, Zhiguo; Guo, Weimin; Chen, Mingxue; Liu, Shuyun [Beijing Key Lab of Regenerative Medicine in Orthopaedics, Institute of Orthopaedics, Chinese PLA General Hospital, Beijing 100853 (China); Key Laboratory of Musculoskeletal Trauma & War Injuries, Institute of Orthopaedics, Chinese PLA General Hospital, Beijing 100853 (China); Xi, Tingfei, E-mail: tingfeixi@163.com [Center for Biomedical Material and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Shenzhen Institute, Peking University, Shenzhen 518057 (China); Guo, Quanyi, E-mail: doctorguo_301@163.com [Beijing Key Lab of Regenerative Medicine in Orthopaedics, Institute of Orthopaedics, Chinese PLA General Hospital, Beijing 100853 (China); Key Laboratory of Musculoskeletal Trauma & War Injuries, Institute of Orthopaedics, Chinese PLA General Hospital, Beijing 100853 (China)

2017-02-01

The objectives of this study were to fabricate porous scaffolds using decellularized meniscus, and to explore a preferable crosslinking condition to enhance mechanical properties of scaffolds. Moreover, the microstructure, porosity, biodegradation and cytotoxicity were also evaluated. EDAC or GTA in different concentration was used to crosslink scaffolds. FTIR demonstrated functional groups change in crosslinking process. SEM photography showed that crosslinked scaffolds had blurry edges, which resulted scaffolds crosslinked by 1.2 mol/l EDAC had smaller porosity than other groups. The structure change enhanced antidegradation property. After immersing in enzyme solution for 96 h, scaffolds crosslinked by GTA and EDAC could maintain their mass > 70% and 80%. Most importantly, mechanical properties of crosslinked scaffolds were also improved. Uncrosslinked Scaffolds had only 0.49 kPa in compression modulus and 12.81 kPa in tensile modulus. The compression and tensile modulus of scaffolds crosslinked by 1.0% GTA were 1.42 and 567.44 kPa respectively. The same value of scaffolds crosslinked by 1.2 mol/l EDAC were 1.49 and 532.50 kPa. Scaffolds crosslinked by 1.0% and 2.5% GTA were toxic to cells, while EDAC groups showed no cytotoxicity. Chondrocytes could proliferate and infiltrate within scaffolds after seeding. Overall, 1.2 mol/l EDAC was a preferable crosslinking condition. - Highlights: • Porous meniscus scaffolds were fabricated using decellularized meniscus tissue. • Mechanical properties of meniscus scaffolds were enhanced by chemical crosslinking. • The crosslinked scaffold showed enhanced anti-degradation properties. • Chondrocytes could infiltrate and proliferate within crosslinked scaffolds.

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

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Gustavo A. Nari

2013-03-01

Full Text Available Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy and biochemical (DNA quantification studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde's staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson's capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusions: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

Influence of pH on extracellular matrix preservation during lung decellularization.

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Tsuchiya, Tomoshi; Balestrini, Jenna L; Mendez, Julio; Calle, Elizabeth A; Zhao, Liping; Niklason, Laura E

2014-12-01

The creation of decellularized organs for use in regenerative medicine requires the preservation of the organ extracellular matrix (ECM) as a means to provide critical cues for differentiation and migration of cells that are seeded onto the organ scaffold. The purpose of this study was to assess the influence of varying pH levels on the preservation of key ECM components during the decellularization of rat lungs. Herein, we show that the pH of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-based decellularization solution influences ECM retention, cell removal, and also the potential for host response upon implantation of acellular lung tissue. The preservation of ECM components, including elastin, fibronectin, and laminin, were better retained in the lower pH conditions that were tested (pH ranges tested: 8, 10, 12); glycosaminoglycans were preserved to a higher extent in the lower pH groups as well. The DNA content following decellularization of the rat lung was inversely correlated with the pH of the decellularization solution. Despite detectible levels of cyotoskeletal proteins and significant residual DNA, tissues decellularized at pH 8 demonstrated the greatest tissue architecture maintenance and the least induction of host response of all acellular conditions. These results highlight the effect of pH on the results obtained by organ decellularization and suggest that altering the pH of the solutions used for decellularization may influence the ability of cells to properly differentiate and home to appropriate locations within the scaffold, based on the preservation of key ECM components and implantation results.

Characterization of porcine eyes based on autofluorescence lifetime imaging

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Batista, Ana; Breunig, Hans Georg; Uchugonova, Aisada; Morgado, António Miguel; König, Karsten

2015-03-01

Multiphoton microscopy is a non-invasive imaging technique with ideal characteristics for biological applications. In this study, we propose to characterize three major structures of the porcine eye, the cornea, crystalline lens, and retina using two-photon excitation fluorescence lifetime imaging microscopy (2PE-FLIM). Samples were imaged using a laser-scanning microscope, consisting of a broadband sub-15 femtosecond (fs) near-infrared laser. Signal detection was performed using a 16-channel photomultiplier tube (PMT) detector (PML-16PMT). Therefore, spectral analysis of the fluorescence lifetime data was possible. To ensure a correct spectral analysis of the autofluorescence lifetime data, the spectra of the individual endogenous fluorophores were acquired with the 16-channel PMT and with a spectrometer. All experiments were performed within 12h of the porcine eye enucleation. We were able to image the cornea, crystalline lens, and retina at multiple depths. Discrimination of each structure based on their autofluorescence intensity and lifetimes was possible. Furthermore, discrimination between different layers of the same structure was also possible. To the best of our knowledge, this was the first time that 2PE-FLIM was used for porcine lens imaging and layer discrimination. With this study we further demonstrated the feasibility of 2PE-FLIM to image and differentiate three of the main components of the eye and its potential as an ophthalmologic technique.

Obtaining liver tridimensional scaffold through the decellularization of rabbit whole liver in 24 hours

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Federico, Schliamser; Ayelen, Rinaldi; Romina, Comin; Alba Nelly, Borchert; Adrian, Nari Gustavo; Alicia, Salvatierra Nancy; Mariana Paula, Cid

2016-04-01

In the present work, we development a new protocol for liver decellularization in which the hole decellularization was reached over 24 h. Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrixin which the time period involved didn't pass 24 h. Methods: The decellularization is obtained through the use of water and SDS (0,1-0,3 %), after freezing at -80 degrees, is the best alternative of different physical and/or chemical mechanisms to break down organ cells and leave only the extracellular matriz. After 24 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy) and biochemical (DNA quantification) studies. Results: the decellularization process was verified by macroscopic observation of the organ using microscopic observation corroborated the macroscopic results, with the hematoxylin-eosin and Masson staining showing no cells or nuclear material. In addition, the DNA quantification was less than 10% in the decellularized liver compared to control. Finally,the time taken to develop the decellularization protocol was less than 24 hours.

Obtaining liver tridimensional scaffold through the decellularization of rabbit whole liver in 24 hours

International Nuclear Information System (INIS)

Federico, Schliamser; Ayelen, Rinaldi; Romina, Comin; Alicia, Salvatierra Nancy; Paula, Cid Mariana; Nelly, Borchert Alba; Adrian, Nari Gustavo

2016-01-01

In the present work, we development a new protocol for liver decellularization in which the hole decellularization was reached over 24 h. Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrixin which the time period involved didn't pass 24 h. Methods: The decellularization is obtained through the use of water and SDS (0,1-0,3 %), after freezing at -80 degrees, is the best alternative of different physical and/or chemical mechanisms to break down organ cells and leave only the extracellular matriz. After 24 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy) and biochemical (DNA quantification) studies. Results: the decellularization process was verified by macroscopic observation of the organ using microscopic observation corroborated the macroscopic results, with the hematoxylin-eosin and Masson staining showing no cells or nuclear material. In addition, the DNA quantification was less than 10% in the decellularized liver compared to control. Finally,the time taken to develop the decellularization protocol was less than 24 hours. (paper)

Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction

International Nuclear Information System (INIS)

Utomo, Lizette; Pleumeekers, Mieke M; Van Osch, Gerjo J V M; Nimeskern, Luc; Stok, Kathryn S; Nürnberger, Sylvia; Hildner, Florian

2015-01-01

Scaffolds are widely used to reconstruct cartilage. Yet, the fabrication of a scaffold with a highly organized microenvironment that closely resembles native cartilage remains a major challenge. Scaffolds derived from acellular extracellular matrices are able to provide such a microenvironment. Currently, no report specifically on decellularization of full thickness ear cartilage has been published. In this study, decellularized ear cartilage scaffolds were prepared and extensively characterized. Cartilage decellularization was optimized to remove cells and cell remnants from elastic cartilage. Following removal of nuclear material, the obtained scaffolds retained their native collagen and elastin contents as well as their architecture and shape. High magnification scanning electron microscopy showed no obvious difference in matrix density after decellularization. However, glycosaminoglycan content was significantly reduced, resulting in a loss of viscoelastic properties. Additionally, in contact with the scaffolds, human bone-marrow-derived mesenchymal stem cells remained viable and are able to differentiate toward the chondrogenic lineage when cultured in vitro. These results, including the ability to decellularize whole human ears, highlight the clinical potential of decellularization as an improved cartilage reconstruction strategy. (paper)

Pressure Shift Freezing as Potential Alternative for Generation of Decellularized Scaffolds

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S. Eichhorn

2013-01-01

Full Text Available Background. Protocols using chemical reagents for scaffold decellularization can cause changes in the properties of the matrix, depending on the type of tissue and the chemical reagent. Technologies using physical techniques may be possible alternatives for the production grafts with potential superior matrix characteristics. Material and Methods. We tested four different technologies for scaffold decellularization. Group 1: high hydrostatic pressure (HHP, 1 GPa; Group 2: pressure shift freezing (PSF; Group 3: pulsed electric fields (PEF; Group 4: control group: detergent (SDS. The degree of decellularization was assessed by histological analysis and the measurement of residual DNA. Results. Tissue treated with PSF showed a decellularization with a penetration depth (PD of 1.5 mm and residual DNA content of . HHD treatment caused a PD of 0.2 mm with a residual DNA content of . PD in PEF was 0.5 mm, and the residual DNA content was . In the SDS group, PD was found to be 5 mm, and the DNA content was determined at . Conclusion. PSF showed promising results as a possible technique for scaffold decellularization. The penetration depth of PSF has to be optimized, and the mechanical as well as the biological characteristics of decellularized grafts have to be evaluated.

Decellularized Swine Dental Pulp as a Bioscaffold for Pulp Regeneration.

Science.gov (United States)

Hu, Lei; Gao, Zhenhua; Xu, Junji; Zhu, Zhao; Fan, Zhipeng; Zhang, Chunmei; Wang, Jinsong; Wang, Songlin

2017-01-01

Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM) is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental pulp ECM from swine and evaluated whether it could mediate pulp regeneration. Dental pulps were acquired from the mandible anterior teeth of swine 12 months of age and decellularized with 10% sodium dodecyl sulfate (SDS) combined with Triton X-100. Pulp regeneration was conducted by seeding human dental pulp stem cells into decellularized pulp and transplanted subcutaneously into nude mice for 8 weeks. The decellularized pulp demonstrated preserved natural shape and structure without any cellular components. Histological analysis showed excellent ECM preservation and pulp-like tissue, and newly formed mineralized tissues were regenerated after being transplanted in vivo. In conclusion, decellularized swine dental pulp maintains ECM components favoring stem cell proliferation and differentiation, thus representing a suitable scaffold for improving clinical outcomes and functions of teeth with dental pulp diseases.

Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization

Energy Technology Data Exchange (ETDEWEB)

Giraldo-Gomez, D.M., E-mail: davidmauro2008@gmail.com [Posgrado en Ciencia e Ingeniería de Materiales, Universidad Nacional Autónoma de México (UNAM), Unidad de Posgrado Edificio “C” 1er Piso, Circuito de Posgrados, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D. F., México (Mexico); Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); Leon-Mancilla, B. [Departamento de Cirugía, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Edificio “D” Planta Baja, Circuito Interior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); Del Prado-Audelo, M.L. [Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), Circuito Exterior, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, C.P. 04510, México D.F., México (Mexico); and others

2016-02-01

There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15 consecutive cycles were performed over 32 days. Protocol B (a variation of Protocol A) — EDTA being added — with 15 consecutive cycles performed over 60 days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5 consecutive cycles being performed over 10 days. For the three protocols, hematoxylin–eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix

Influence of quercetin and nanohydroxyapatite modifications of decellularized goat-lung scaffold for bone regeneration

Energy Technology Data Exchange (ETDEWEB)

Gupta, Sweta K. [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, 247667 (India); Kumar, Ritesh [Center for Computational Biology, University of Kansas, Kansas 66045 (United States); Mishra, Narayan C., E-mail: mishrawise@gmail.com [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, 247667 (India)

2017-02-01

In the present study, goat-lung scaffold was fabricated by decellularization of lung tissue and verified for complete cell removal by DNA quantification, DAPI and H&E staining. The scaffold was then modified by crosslinking with quercetin and nanohydroxyapatite (nHAp), and characterized to evaluate the suitability of quercetin-crosslinked nHAp-modified scaffold for regeneration of bone tissue. The crosslinking chemistry between quercetin and decellularized scaffold was established theoretically by AutoDock Vina program (in silico docking study), which predicted multiple intermolecular hydrogen bonding interactions between quercetin and decellularized scaffold, and FTIR spectroscopy analysis also proved the same. From MTT assay and SEM studies, it was found that the quercetin-crosslinked nHAp-modified decellularized scaffold encouraged better growth and proliferation of bone-marrow derived mesenchymal stem cells (BMMSCs) in comparison to unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold. Alkaline Phosphatase (ALP) assay results showed highest expression of ALP over quercetin-crosslinked nHAp-modified scaffold among all the tested scaffolds (unmodified decellularized scaffold, quercetin-crosslinked decellularized scaffold and nHAp-modified decellularized scaffold) − indicating that quercetin and nHAp is very much efficient in stimulating the differentiation of BMMSCs into osteoblast cells. Alizarin red test quantified in vitro mineralization (calcium deposits), and increased expression of alizarin red over quercetin-crosslinked nHAp-modified scaffold indicating better stimulation of osteogenesis in BMMSCs. The above findings suggest that quercetin-crosslinked nHAp-modified decellularized goat-lung scaffold provides biomimetic bone-like microenvironment for BMMSCs to differentiate into osteoblast and could be applied as a potential promising biomaterial for bone regeneration. - Highlights: �

Decellularized allogeneic heart valves demonstrate self-regeneration potential after a long-term preclinical evaluation.

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Laura Iop

Full Text Available Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT reconstruction in Vietnamese Pigs (n = 11 with 6 (n = 5 and 15 (n = 6 follow-up months. Repositioned native valves (n = 2 for each time were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly

Decellularization of Human Internal Mammary Artery: Biomechanical Properties and Histopathological Evaluation.

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Kajbafzadeh, Abdol-Mohammad; Khorramirouz, Reza; Kameli, Seyede Maryam; Hashemi, Javad; Bagheri, Amin

2017-01-01

This study undertook to create small-diameter vascular grafts and assess their structure and mechanical properties to withstand arterial implantation. Twenty samples of intact human internal mammary arteries (IMAs) were collected and decellularized using detergent-based methods. To evaluate residual cellular and extracellular matrix (ECM) components, histological analysis was performed. Moreover, collagen typing and ECM structure were analyzed by Picrosirius red and Movat's pentachrome staining. Scanning electron microscopy was also applied to assess microarchitecture of both endothelial and adventitial surfaces of native and decellularized arterial samples. Furthermore, mechanical tests were performed to evaluate the rigidity and suture strength of the arteries. Human IMAs were completely decellularized in all three segments (proximal, middle, and distal). ECM proteins such as collagen and elastic fibers were efficiently preserved and no structural distortion in intima, media, and adventitial surfaces was observed. The parameters of the mechanical tests revealed no significant differences in the mechanical properties of decellularized arteries in comparison to native arteries with considerable strength, suture retention, and stress relaxation (Young's modulus [MPa] = 0.22 ± 0.023 [native] and 0.22 ± 0.015 [acellular]; and suture strength 0.56 ± 0.19 [native] vs. 0.56 ± 0.12 [acellular], respectively). Decellularized IMA represents a potential arterial scaffold as an alternative to autologous grafts for future arterial bypass surgeries. By this technique, microarchitecture and mechanical integrity of decellularized arteries were considerably similar to native arteries. The goal of this study was to introduce an efficient method for complete decellularization of human IMA and evaluate the ECM and biomechanical properties.

Decellularized Swine Dental Pulp as a Bioscaffold for Pulp Regeneration

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Lei Hu

2017-01-01

Full Text Available Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental pulp ECM from swine and evaluated whether it could mediate pulp regeneration. Dental pulps were acquired from the mandible anterior teeth of swine 12 months of age and decellularized with 10% sodium dodecyl sulfate (SDS combined with Triton X-100. Pulp regeneration was conducted by seeding human dental pulp stem cells into decellularized pulp and transplanted subcutaneously into nude mice for 8 weeks. The decellularized pulp demonstrated preserved natural shape and structure without any cellular components. Histological analysis showed excellent ECM preservation and pulp-like tissue, and newly formed mineralized tissues were regenerated after being transplanted in vivo. In conclusion, decellularized swine dental pulp maintains ECM components favoring stem cell proliferation and differentiation, thus representing a suitable scaffold for improving clinical outcomes and functions of teeth with dental pulp diseases.

Mechanical compliance and immunological compatibility of fixative-free decellularized/cryopreserved human pericardium.

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Maria Cristina Vinci

Full Text Available BACKGROUND: The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue. PRINCIPAL FINDINGS: The aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice. CONCLUSIONS: Our results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.

Penetration of mucoadhesive chitosan-dextran sulfate nanoparticles into the porcine cornea.

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Chaiyasan, Wanachat; Praputbut, Sakonwun; Kompella, Uday B; Srinivas, Sangly P; Tiyaboonchai, Waree

2017-01-01

Topical application of drugs to the eyes suffers from poor bioavailability at the ocular surface and in the anterior chamber. This is due to rapid clearance of the drug because of tear secretion and outflow. This study has investigated mucoadhesive and penetration characteristics of chitosan-dextran sulfate nanoparticles (CDNs), prepared by polyelectrolyte complexation technique, following topical administration to the ocular surface. Topical FITC-labeled CDNs (FCDNs; mean size of 400nm and a surface charge of +48mV) were retained on the porcine ocular surface for more than 4h. Topical FCDNs were partially endocytosed into porcine corneal epithelial cells via a clathrin-dependent pathway. After 6h of topical FCDNs, particles accumulated in the corneal epithelium but not found in the corneal stroma. When epithelium was removed, FCDNs penetrated the stroma. Thus, CDNs are potentially useful for drug/gene delivery to the ocular surface and to stroma when epithelium is damaged. Copyright © 2016 Elsevier B.V. All rights reserved.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

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Gershlak, Joshua R; Hernandez, Sarah; Fontana, Gianluca; Perreault, Luke R; Hansen, Katrina J; Larson, Sara A; Binder, Bernard Y K; Dolivo, David M; Yang, Tianhong; Dominko, Tanja; Rolle, Marsha W; Weathers, Pamela J; Medina-Bolivar, Fabricio; Cramer, Carole L; Murphy, William L; Gaudette, Glenn R

2017-05-01

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

[Experimental study on co-culture of human fibroblasts on decellularized Achilles tendon].

Science.gov (United States)

Wang, Zhibing; Zhang, Xia; Guo, Xinyu; Qin, Chuan

2013-07-01

To investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits (5-month-old; weighing, 4-5 kg). The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate (SDS), and then gross observation, histological examination, and scanning electron microscope (SEM) observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. After decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young's elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group (P Achilles tendons group and cell-scaffold composite group (P > 0.05). There was no significant difference in elongation at break among 3 groups (P > 0.05). The decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.

Bacterial transmission from contact lenses to porcine corneas : An ex vivo study

NARCIS (Netherlands)

Vermeltfoort, Pit B. J.; van Kooten, Theo G.; Bruinsma, Gerda .; Hooymans, Anneke M. M.; van der Mei, Henny C.; Busscher, Henk J.

PURPOSE. To quantify the transmission to ex vivo porcine eyes of Staphylococcus aureus 835 and Pseudomonas aeruginosa 3 from three types of contact lenses - one daily wear and two extended wear - differing in hydrophobicity and roughness. METHODS. One daily wear lens (etafilcon) and two

The impact of detergents on the tissue decellularization process: A ToF-SIMS study.

Science.gov (United States)

White, Lisa J; Taylor, Adam J; Faulk, Denver M; Keane, Timothy J; Saldin, Lindsey T; Reing, Janet E; Swinehart, Ilea T; Turner, Neill J; Ratner, Buddy D; Badylak, Stephen F

2017-03-01

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy. We report here on the use of a highly sensitive analytical technique, time of flight secondary ion mass spectroscopy (ToF-SIMS) to characterize detergent decellularized scaffolds. ToF-SIMS detected cellular remnants and residual detergent fragments; increased intensity of the detergent fragments correlated with adverse cell matrix interactions. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and matrix surface ligand landscape. This study also demonstrates the power of ToF-SIMS for the characterization of decellularized scaffolds and capability for assessment of decellularization efficacy. Future use of biologic scaffolds in clinical tissue reconstruction will benefit from the fundamental results described in this work. Copyright © 2016 Acta

In vivo immunogenicity of bovine bone removed by a novel decellularization protocol based on supercritical carbon dioxide.

Science.gov (United States)

You, Ling; Weikang, Xu; Lifeng, Yang; Changyan, Liang; Yongliang, Lin; Xiaohui, Wei; Bin, Xu

2018-05-04

Trauma or infections associated critical bone defects lead to a huge economic burden in the healthcare system worldwide. Recent advances in tissue engineering have led to potential new strategies for the repair, replacement, and regeneration of bone defects, especially in biomaterials and decellularization protocols from xenogenic tissues. However, the complexity in bone structure and mechanical environment limits the synthesis of artificial bone with biomaterials. Thus, the purpose of our study is to develop a natural bone scaffold with great immunocompatibility. We combined decellularization techniques base on SC-CO 2 to decellularize bovine bone. In order to study the immune response of mice to materials, the histology, spleen index, immune cells contents and in vitro proliferative performance, cytokine and immunoglobulin light chain expression of mice were characterized. Compared with the fresh bone group, the immune responses of decellularized group were significantly reduced. In conclusion, decellularization via this method can achieve a decellularized scaffold with great immunocompatibility. Our findings suggest the potential of using decellularized BB as a scaffold for bone bioengineering.

Determination of permeability coefficients of ophthalmic drugs through different layers of porcine, rabbit and bovine eyes.

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Loch, Christian; Zakelj, Simon; Kristl, Albin; Nagel, Stefan; Guthoff, Rudolf; Weitschies, Werner; Seidlitz, Anne

2012-08-30

To treat ophthalmic diseases like glaucoma or inflammatory disorders topically applied ophthalmic formulations such as eye drops are usually used. In addition, novel ophthalmic implants releasing drug substances locally into different parts of the eye are available today. In the work presented here, the permeability coefficients of selected drugs (ciprofloxacin hydrochloride, lidocaine hydrochloride, timolol maleate) for ophthalmic tissues were determined using side-by-side diffusion chambers (so-called Ussing chambers). Sclera, conjunctiva, cornea, choroidea-retina-complex and a complex of conjunctiva-sclera-choroidea-retina were excised from fresh porcine, rabbit and bovine eyes. In the porcine eye tissues the highest P(app) values were obtained for conjunctiva with the exception of lidocaine. Therefore, it can be estimated that a certain amount of drug diffuses or is transported through conjunctiva after application. The P(app) values for sclera were also higher than those for cornea and even more, the surface area of sclera which is available for drug absorption is much larger than that of cornea when applying an implant. The obtained permeability coefficients for sclera and conjunctiva indicate that the administration of periocular implants can be an alternative to topically applied formulations. The complexes of the tissues were a significantly (p<0.01) stronger barrier to the investigated substances than the separated tissues. Distinct differences in permeability coefficients between the investigated animal tissues were observed. Overall the highest P(app) values for all mounted tissues were obtained with the rabbit, followed by porcine and bovine eyes. Because of these distinct interspecies differences one must be very careful when selecting the proper animal model for the permeability experiments. Copyright © 2012 Elsevier B.V. All rights reserved.

Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

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Andrea Porzionato

2015-07-01

Full Text Available Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.

Decellularized Rat Lung Scaffolds Using Sodium Lauryl Ether Sulfate for Tissue Engineering.

Science.gov (United States)

Ma, Jinhui; Ju, Zhihai; Yu, Jie; Qiao, Yeru; Hou, Chenwei; Wang, Chen; Hei, Feilong

Perfusion decellularization with detergents is effective to maintain the architecture and proteins of extracellular matrix (ECM) for use in the field of lung tissue engineering (LTE). However, it is unclear which detergent is ideal to produce an acellular lung scaffold. In this study, we obtained two decellularized rat lung scaffolds using a novel detergent sodium lauryl ether sulfate (SLES) and a conventional detergent sodium dodecyl sulfate (SDS). Both decellularized lung scaffolds were assessed by histology, immunohistochemistry, scanning electron microscopy, DNA quantification, sulfated glycosaminoglycans (GAGs) quantification and western blot. Subsequently, the scaffolds were implanted subcutaneously in rats for 6 weeks and were evaluated via hematoxylin and eosin staining and Masson staining. Results indicated that SLES was effective to remove cells; moreover, lungs decellularized with SLES showed better preservation of sulfated GAGs, lung architecture, and ECM proteins than SDS. After 6 weeks, SLES scaffolds demonstrated a significantly greater potential for cell infiltration and blood vessel formation compared with SDS scaffolds. Taken together, we conclude that SLES is a promising detergent to produce an acellular scaffold using LTE for eventual transplantation.

Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis.

Science.gov (United States)

Seo, Yoojin; Jung, Youngmee; Kim, Soo Hyun

2018-02-01

Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO 2 -EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO 2 reactor and decellularized at 37 °C and 350 bar. After scCO 2 -EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO 2 -EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO 2 -EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for

Decellularization of Human Nasal Septal Cartilage for the Novel Filler Material of Vocal Fold Augmentation.

Science.gov (United States)

Kang, Dae-Woon; Shin, Sung-Chan; Jang, Jeon-Yeob; Park, Hee-Young; Lee, Jin-Choon; Wang, Soo-Geun; Lee, Byung-Joo

2017-01-01

The clinical application of allogenic and/or xenogenic cartilage for vocal fold augmentation requires to remove the antigenic cellular component. The objective of this study was to assess the effect of cartilage decellularization and determine the change in immunogenicity after detergent treatment in human nasal septal cartilage flakes made by the freezing and grinding method. Human nasal septal cartilages were obtained from surgical cases. The harvested cartilages were treated by the freezing and grinding technique. The obtained cartilage flakes were treated with 1% Triton X-100 or 2% sodium dodecyl sulfate (SDS) for decellularization of the cartilage flakes. Hematoxylin and eosin stain (H&E stain), surface electric microscopy, immunohistochemical stain for major histocompatibility complex I and II, and ELISA for DNA contents were performed to assess the effect of cartilage decellularization after detergent treatment. A total of 10 nasal septal cartilages were obtained from surgical cases. After detergent treatment, the average size of the cartilage flakes was significantly decreased. With H&E staining, the cell nuclei of decellularized cartilage flakes were not observed. The expression of major histocompatibility complex (MHC)-I and II antigens was not identified in the decellularized cartilage flakes after treatment with detergent. DNA content was removed almost entirely from the decellularized cartilage flakes. Treatment with 2% SDS or 1% Triton X-100 for 1 hour appears to be a promising method for decellularization of human nasal septal cartilage for vocal fold augmentation. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Decellularized Tissue and Cell-Derived Extracellular Matrices as Scaffolds for Orthopaedic Tissue Engineering

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Cheng, Christina W.; Solorio, Loran D.; Alsberg, Eben

2014-01-01

The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic. PMID:24417915

Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

KAUST Repository

Gerhardt, Lutz Christian

2012-09-11

In this study, the in vivo recellularization and neovascularization of nanosized bioactive glass (n-BG)-coated decellu-larized trabecular bone scaffolds were studied in a rat model and quantified using stereological analyses. Based on the highest amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm 2), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating density of 0.263 mg/cm2, human fibroblasts produced 4.3 times more VEGF than on uncoated controls. After 8 weeks of implantation in Sprague-Dawley rats, both uncoated and n-BG-coated samples were well infiltrated with newly formed tissue (47-48%) and blood vessels (3-4%). No significant differences were found in cellularization and vascularization between uncoated bone scaffolds and n-BG-coated scaffolds. This finding indicates that the decellularized bone itself may exhibit growth-promoting properties induced by the highly interconnected pore microarchitecture and/or proteins left behind on decellularized scaffolds. Even if we did not find proangiogenic effects in n-BG-coated bone scaffolds, a bioactive coating is considered to be beneficial to impart osteoinductive and osteoconductive properties to decellularized bone. n-BG-coated bone grafts have thus high clinical potential for the regeneration of complex tissue defects given their ability for recellularization and neovascularization. © 2012 Wiley Periodicals, Inc.

Biophysico-functional compatibility of Seoul National University (SNU) miniature pig cornea as xenocorneal graft for the use of human clinical trial.

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Kim, Dong Hyun; Kim, Jaeyoung; Jeong, Hyun Jeong; Lee, Hyun Ju; Kim, Mee Kum; Wee, Won Ryang

2016-05-01

Xenocorneal transplantation is one of the solutions for shortage of donor cornea, and remarkable advances have been made in pig-to-rhesus studies from the immunological perspective. Most successful preclinical trials have been carried out with corneas of the Seoul National University (SNU) miniature pig (SNU pig, genetically unmodified) as donor tissues; however, there has been no biophysico-functional evaluation of the SNU pig cornea as a substitute for human cornea. The purpose of this study was to investigate the biophysical and functional compatibility of SNU pig cornea for use in human clinical trials. Ninety-three eyeballs obtained from 51 SNU pigs were used to evaluate the physical properties and changes in porcine corneal endothelial cells (PCECs) depending on preservation time and storage condition before surgery, proliferative and functional characteristics of PCECs, and the microbiologic safety of porcine cornea. Corneal diameters and curvatures, axial length, anterior chamber depth, and central corneal thickness were measured and compared with previously reported human data. Corneal endothelial cell density (ECD) was serially measured with a confocal microscope during 7 days of preservation in the same storage solution used for human corneas. Corneal endothelial cell proliferation and immunofluorescence staining of Na- and K-dependent ATPase in PCECs were evaluated after 7 days of preservation. The corneoscleral rims of SNU pigs were cultured for gram-positive bacteria, gram-negative bacteria, and fungi to evaluate their microbiological safety. Corneal diameter and thickness in SNU pigs was larger than human and corneal curvature was flatter; however, they were within surgically operable ranges. Mean ECD (day 0) and ECD loss after 7 days of preservation were 2625 ± 81 cells/mm(2) and 7.60 ± 1.53%, respectively, which is comparable to human ECD and ECD loss in the same conditions. The ECD of SNU pigs was inversely decreased with aging (R(2) = 0.4034, P

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

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Amanda J Sutherland

Full Text Available Extracellular matrix (ECM-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs were cultured in cell pellets containing cells only (control, chondrogenic differentiation medium (TGF-β, chemically decellularized cartilage particles (DCC, or physically devitalized cartilage particles (DVC. The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

International Nuclear Information System (INIS)

Farhat, Walid A; Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman; Sherman, Christopher; Derwin, Kathleen

2008-01-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

Science.gov (United States)

Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

Energy Technology Data Exchange (ETDEWEB)

Farhat, Walid A [Department of Surgery, Division of Urology, University of Toronto and Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman [Department of Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Sherman, Christopher [Department of Anatomic Pathology, Sunnybrook and Women' s College Health Sciences Centre, Toronto, ON (Canada); Derwin, Kathleen [Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, OH (United States)], E-mail: walid.farhat@sickkids.ca

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Construction of Thymus Organoids from Decellularized Thymus Scaffolds.

Science.gov (United States)

Tajima, Asako; Pradhan, Isha; Geng, Xuehui; Trucco, Massimo; Fan, Yong

2016-10-12

One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progress has been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma. Here, we describe the construction of functional thymus organoids from decellularized thymus scaffolds repopulated with isolated TECs. Thymus decellularization was achieved by freeze-thaw cycles to induce intracellular ice crystal formation, followed by detergent-induced cell lysis. Cellular debris was removed with extensive wash. The decellularized thymus scaffolds can largely retain the 3D extracellular matrix (ECM) microenvironment that can support the recolonization of TECs. When transplanted into athymic nude mice, the reconstructed thymus organoids can effectively promote the homing of bone marrow-derived lymphocyte progenitors and support the development of a diverse and functional T cell repertoire. Bioengineering of thymus organoids can be a promising approach to rejuvenate/modulate the function of T-cell mediated adaptive immunity in regenerative medicine.

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

OpenAIRE

Nari, Gustavo A.; Cid, Mariana; Comín, Romina; Reyna, Laura; Juri, Gustavo; Taborda, Ricardo; Salvatierra, Nancy A.

2013-01-01

Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemica...

Modification of Rat Lung Decellularization Protocol Based on Dynamic Conductometry of Working Solution.

Science.gov (United States)

Kuevda, E V; Gubareva, E A; Gumenyuk, I S; Sotnichenko, A S; Gilevich, I V; Nakokhov, R Z; Rusinova, T V; Yudina, T G; Red'ko, A N; Alekseenko, S N

2017-03-01

We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA.

Porous decellularized tissue engineered hypertrophic cartilage as a scaffold for large bone defect healing.

Science.gov (United States)

Cunniffe, Gráinne M; Vinardell, Tatiana; Murphy, J Mary; Thompson, Emmet M; Matsiko, Amos; O'Brien, Fergal J; Kelly, Daniel J

2015-09-01

Clinical translation of tissue engineered therapeutics is hampered by the significant logistical and regulatory challenges associated with such products, prompting increased interest in the use of decellularized extracellular matrix (ECM) to enhance endogenous regeneration. Most bones develop and heal by endochondral ossification, the replacement of a hypertrophic cartilaginous intermediary with bone. The hypothesis of this study is that a porous scaffold derived from decellularized tissue engineered hypertrophic cartilage will retain the necessary signals to instruct host cells to accelerate endogenous bone regeneration. Cartilage tissue (CT) and hypertrophic cartilage tissue (HT) were engineered using human bone marrow derived mesenchymal stem cells, decellularized and the remaining ECM was freeze-dried to generate porous scaffolds. When implanted subcutaneously in nude mice, only the decellularized HT-derived scaffolds were found to induce vascularization and de novo mineral accumulation. Furthermore, when implanted into critically-sized femoral defects, full bridging was observed in half of the defects treated with HT scaffolds, while no evidence of such bridging was found in empty controls. Host cells which had migrated throughout the scaffold were capable of producing new bone tissue, in contrast to fibrous tissue formation within empty controls. These results demonstrate the capacity of decellularized engineered tissues as 'off-the-shelf' implants to promote tissue regeneration. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Generation of Femtosecond Laser-Cut Decellularized Corneal Lenticule Using Hypotonic Trypsin-EDTA Solution for Corneal Tissue Engineering

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Man-Il Huh

2018-01-01

Full Text Available Purpose. To establish an optimized and standardized protocol for the development of optimal scaffold for bioengineering corneal substitutes, we used femtosecond laser to process human corneal tissue into stromal lenticules and studied to find the most efficient decellularization method among various reagents with different tonicities. Methods. The decellularization efficacy of several agents (0.1%, 0.25%, and 0.5% of Triton X-100, SDS, and trypsin-EDTA (TE, resp. with different tonicities was evaluated. Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and glycosaminoglycan (GAG contents, recellularization efficacy, and biocompatibilities. Results. 0.5% SDS in hypertonic and isotonic buffer, 0.25% TE in hypotonic buffer, and 0.5% TE in all tonicities completely decellularized the corneal lenticules. Of the protocols, decellularization with hypotonic 0.25 and 0.5% TE showed the lowest DNA contents, while the GAG content was the highest. Furthermore, the recellularization efficacy of the hypotonic TE method was better than that of the SDS-based method. Hypotonic TE-treated decellularized corneal lenticules (DCLs were sufficiently transparent and biocompatible. Conclusion. We generated an ideal protocol for DCLs using a novel method. Furthermore, it is possible to create a scaffold using a bioengineered corneal substitute.

Decellularized Bovine Articular Cartilage Matrix Reinforced by Carboxylated-SWCNT for Tissue Engineering Application

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Zari Majidi Mohammadie

2018-01-01

Full Text Available ABSTRACT Nanotubes with their unique properties have diversified mechanical and biological applications. Due to similarity of dimensions with extracellular matrix (ECM elements, these materials are used in designing scaffolds. In this research, Carboxylated Single-Wall Carbon Nanotubes in optimization of decellularized scaffold of bovine articular cartilage was used. At first, the articular cartilage was decellularized. Then the scaffolds were analyzed in: (i decellularized scaffolds, and (ii scaffolds plunged into homogenous suspension of nanotubes in distilled water, were smeared with Carboxylated-SWCNT. The tissue rings derived from the rabbit's ear were assembled with reinforced scaffolds and they were placed in a culture media for 15 days. The scaffolds in two groups and the assembled scaffolds underwent histologic and electron microscopy. Scanning electron microscopy showed that the structure of ECM of articular cartilage has been maintained well after decellularization. Fourier transform infrared analysis showed that the contents of ECM have not been changed under treatment process. Atomic force microscopy analysis showed the difference in surface topography and roughness of group (ii scaffolds in comparison with group (i. Transmission electron microscopy studies showed the Carboxylated-SWCNT bond with the surface of decellularized scaffold and no penetration of these compounds into the scaffold. The porosity percentage with median rate of 91.04 in group (i scaffolds did not have significant difference with group (ii scaffolds. The electron microscopy observations confirmed migration and penetration of the blastema cells into the group (ii assembled scaffolds. This research presents a technique for provision of nanocomposite scaffolds for cartilage engineering applications.

Comparison of endothelial cell density of organ cultured corneas with cornea donor study.

Science.gov (United States)

Campolmi, Nelly; He, Zhiguo; Acquart, Sophie; Trone, Marie-Caroline; Bernard, Aurélien; Gauthier, Anne-Sophie; Garraud, Olivier; Forest, Fabien; Péocʼh, Michel; Gain, Philippe; Thuret, Gilles

2014-06-01

Determination of the endothelial cell density (ECD) by eye banks is paramount in donor cornea qualification. Unbiased measurement avoids wastage and grafts with an increased risk of premature failure. Internal calibration of the counting method is essential, but external validation would add an extra stage in the assessment of reliability. In this respect, data published by the multicenter Cornea Donor Study (CDS) in 2005 is a reference. The aim of the study was to compare ECD determined within a single eye bank, which uses calibrated image analysis software designed for transmitted light microscopy images of organ cultured corneas, with the CDS data determined on specular microscopy images of corneas stored at 4°C. ECD of consecutive corneas retrieved between 2005 and 2013 was determined after exposure to 0.9% NaCl. More than 300 ECs were counted on 3 fields of the central 8 mm. Endothelial cell boundaries were automatically drawn and verified by a skilled technician who performed all necessary corrections. Three thousand fifty-two corneas were analyzed, of which 48.5% donors were >75 years (CDS upper age limit). Between 10 and 75 years, the ECD varied according to donor age exactly in the same manner as in the CDS, but were consistently higher of 100 ± 25 cells per square millimeter (P Atlantic Ocean could be due to (1) differences in counting principles and/or (2) higher shrinkage of the cornea caused by stromal edema in organ culture.

Decellularized Human Dental Pulp as a Scaffold for Regenerative Endodontics.

Science.gov (United States)

Song, J S; Takimoto, K; Jeon, M; Vadakekalam, J; Ruparel, N B; Diogenes, A

2017-06-01

Teeth undergo postnatal organogenesis relatively late in life and only complete full maturation a few years after the crown first erupts in the oral cavity. At this stage, development can be arrested if the tooth organ is damaged by either trauma or caries. Regenerative endodontic procedures (REPs) are a treatment alternative to conventional root canal treatment for immature teeth. These procedures rely on the transfer of apically positioned stem cells, including stem cells of the apical papilla (SCAP), into the root canal system. Although clinical success has been reported for these procedures, the predictability of expected outcomes and the organization of the newly formed tissues are affected by the lack of an available suitable scaffold that mimics the complexity of the dental pulp extracellular matrix (ECM). In this study, we evaluated 3 methods of decellularization of human dental pulp to be used as a potential autograft scaffold. Tooth slices of human healthy extracted third molars were decellularized by 3 different methods. One of the methods generated the maximum observed decellularization with minimal impact on the ECM composition and organization. Furthermore, recellularization of the scaffold supported the proliferation of SCAP throughout the scaffold with differentiation into odontoblast-like cells near the dentinal walls. Thus, this study reports that human dental pulp from healthy extracted teeth can be successfully decellularized, and the resulting scaffold supports the proliferation and differentiation of SCAP. The future application of this form of an autograft in REPs can fulfill a yet unmet need for a suitable scaffold, potentially improving clinical outcomes and ultimately promoting the survival and function of teeth with otherwise poor prognosis.

Fabrication method, structure, mechanical, and biological properties of decellularized extracellular matrix for replacement of wide bone tissue defects.

Science.gov (United States)

Anisimova, N Y; Kiselevsky, M V; Sukhorukova, I V; Shvindina, N V; Shtansky, D V

2015-09-01

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125-130 MPa and the flexural strength of humerus bones was 156 and 145 MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as

Optimization of SDS exposure on preservation of ECM characteristics in whole organ decellularization of rat kidneys.

Science.gov (United States)

He, M; Callanan, A; Lagaras, K; Steele, J A M; Stevens, M M

2017-08-01

Renal transplantation is well established as the optimal form of renal replacement therapy but is restricted by the limited pool of organs available for transplantation. The whole organ decellularisation approach is leading the way for a regenerative medicine solution towards bioengineered organ replacements. However, systematic preoptimization of both decellularization and recellularization parameters is essential prior to any potential clinical application and should be the next stage in the evolution of whole organ decellularization as a potential strategy for bioengineered organ replacements. Here we have systematically assessed two fundamental parameters (concentration and duration of perfusion) with regards to the effects of differing exposure to the most commonly used single decellularizing agent (sodium dodecyl sulphate/SDS) in the perfusion decellularization process for whole rat kidney ECM bioscaffolds, with findings showing improved preservation of both structural and functional components of the whole kidney ECM bioscaffold. Whole kidney bioscaffolds based on our enhanced protocol were successfully recellularized with rat primary renal cells and mesenchymal stromal cells. These findings should be widely applicable to decellularized whole organ bioscaffolds and their optimization in the development of regenerated organ replacements for transplantation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1352-1360, 2017. © 2016 Wiley Periodicals, Inc.

Decellularized ovine arteries as small-diameter vascular grafts

International Nuclear Information System (INIS)

Mancuso, L; Cao, G; Gualerzi, A; Boschetti, F; Loy, F

2014-01-01

Atherosclerosis and its complications still represent the leading cause of death in the developed countries. While autologous blood vessels may be regarded as the best solution for peripheral and coronary bypass, they are unavailable in most patients. Even though tissue engineering techniques are often applied to the development of small-diameter vascular grafts, limiting factors of this approach are represented by the lack of essential extracellular matrix proteins and/or poor biomechanical properties of the scaffolds used. Along these lines, the aim of this study was to develop a decellularization protocol for ovine carotids to be used as suitable small-diameter vascular grafts. Samples were treated either with sodium dodecyl sulphate (SDS) or with Trypsin and Triton X-100; a final nuclease digestion was performed for both protocols. Morphological analyses demonstrate complete removal of nuclei and cellular components in treated vessels, also confirmed by significant reduction in wall thickness and DNA content. Essential extracellular matrix proteins such as collagen, elastin, and fibronectin are well preserved after decellularization. From a mechanical point of view, Trypsin and Triton X-100 treated arteries show elastic modules and compliance comparable to native carotids, whereas the use of SDS makes samples stiffer, with a significant decrease in the compliance mean value and an increase in longitudinal and circumferential Young’s modules. It is demonstrated that the treatment where Trypsin and Triton X-100 are combined guarantees complete decellularization of carotids, with no significant alteration of biomechanical and structural properties, thus preserving a suitable environment for adhesion, proliferation, and migration of cells. (paper)

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique.

Science.gov (United States)

Xu, Yong; Li, Dan; Yin, Zongqi; He, Aijuan; Lin, Miaomiao; Jiang, Gening; Song, Xiao; Hu, Xuefei; Liu, Yi; Wang, Jinpeng; Wang, Xiaoyun; Duan, Liang; Zhou, Guangdong

2017-08-01

Tissue-engineered trachea provides a promising approach for reconstruction of long segmental tracheal defects. However, a lack of ideal biodegradable scaffolds greatly restricts its clinical translation. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration owing to natural tubular structure, cartilage matrix components, and biodegradability. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. To address these problems, a laser micropore technique (LMT) was applied in the current study to modify trachea sample porosity to facilitate decellular treatment and cell ingrowth. Decellularization processing demonstrated that cells in LMT treated samples were more easily removed compared with untreated native trachea. Furthermore, after optimizing the protocols of LMT and decellular treatments, the LMT-treated DTM (LDTM) could retain their original tubular shape with only mild extracellular matrix damage. After seeding with chondrocytes and culture in vitro for 8 weeks, the cell-LDTM constructs formed tubular cartilage with relatively homogenous cell distribution in both micropores and bilateral surfaces. In vivo results further confirmed that the constructs could form mature tubular cartilage with increased DNA and cartilage matrix contents, as well as enhanced mechanical strength, compared with native trachea. Collectively, these results indicate that LDTM is an ideal scaffold for tubular cartilage regeneration and, thus, provides a promising strategy for functional reconstruction of trachea cartilage. Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the

The effect of riboflavin/UVA cross-linking on anti-degeneration and promoting angiogenic capability of decellularized liver matrix.

Science.gov (United States)

Xiang, Junxi; Liu, Peng; Zheng, Xinglong; Dong, Dinghui; Fan, Shujuan; Dong, Jian; Zhang, Xufeng; Liu, Xuemin; Wang, Bo; Lv, Yi

2017-10-01

Weak mechanical property and unstable degradation rate limited the application of decellularized liver matrix in tissue engineering. The aim of this study was to explore a new method for improving the mechanical properties, anti-degeneration and angiogenic capability of decellularized liver matrix. This was achieved by a novel approach using riboflavin/ultraviolet A treatment to induce collagen cross-linking of decellularized matrix. Histological staining and scanning electron microscope showed that the diameter of cross-linked fibers significantly increased compared with the control group. The average peak load and Young's modulus of decellularized matrix were obviously improved after cross-linking. Then we implanted the modified matrix into the rat hepatic injury model to test the anti-degeneration and angiogenic capability of riboflavin/UVA cross-linked decellularized liver scaffolds in vivo. The results indicated that cross-linked scaffolds degrade more slowly than those in the control group. In the experiment group, average microvessel density in the implanted matrix was higher than that in the control group since the first week after implantation. In conclusion, we initiated the method to improve the biomechanical properties of decellularized liver scaffolds by riboflavin/UVA cross-linking, and more importantly, its improvement on anti-degeneration and angiogenesis was identified. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2662-2669, 2017. © 2017 Wiley Periodicals, Inc.

Mesenchymal stem cells can survive on the extracellular matrix-derived decellularized bovine articular cartilage scaffold

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Amin Tavassoli

2015-12-01

Full Text Available Objective (s: The scarcity of articular cartilage defect to repair due to absence of blood vessels and tissue engineering is one of the promising approaches for cartilage regeneration. The objective of this study was to prepare an extracellular matrix derived decellularized bovine articular cartilage scaffold and investigate its interactions with seeded rat bone marrow mesenchymal stem cells (BM-MSCs. Materials and Methods: Bovine articular cartilage that was cut into pieces with 2 mm thickness, were decellularized by combination of physical and chemical methods including snap freeze-thaw and treatment with sodium dodecyl sulfate (SDS. The scaffolds were then seeded with 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (DiI labeled BM-MSCs and cultured for up to two weeks. Results: Histological studies of decellularized bovine articular cartilage showed that using 5 cycles of snap freeze-thaw in liquid nitrogen and treatment with 2.5% SDS for 4 hr led to the best decellularization, while preserving the articular cartilage structure. Adherence and penetration of seeded BM-MSCs on to the scaffold were displayed by histological and florescence examinations and also confirmed by electron microscopy. Conclusion: ECM-derived decellularized articular cartilage scaffold provides a suitable environment to support adhesion and maintenance of cultured BM-MSCs and could be applied to investigate cellular behaviors in this system and may also be useful for studies of cartilage tissue engineering.

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

Science.gov (United States)

Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

Live or Let Die: Epithelial Flap Vitality and Keratocyte Proliferation Following LASEK and Epi-LASIK in Human Donor and Porcine Eyes.

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Angunawela, Romesh I; Winkler von Mohrenfels, Christoph; Kumar, Anupma; O'Brart, David P S; Marshall, John

2011-02-01

To determine the relationship between epithelial flap vitality and stromal keratocyte proliferation following two epithelial refractive techniques: epi-LASIK and laser epithelial keratomileusis (LASEK). Human corneas were maintained in organ culture and underwent standard -6.00-diopter ablation. Rates of stromal keratocyte proliferation were detected 1 week postoperative using a Ki67 antibody specific to proliferating cells. Images were captured with a laser scanning confocal microscope and analyzed by a masked observer. Epithelial flap vitality was determined with propidium iodide using fresh porcine corneas. Epithelial flaps were created with Gebauer Epikeratome epi-LASIK or alcohol-assisted LASEK method. Flaps treated with 100% alcohol and uninjured corneas were used as controls. The number of proliferating keratocytes was greatest at 1 week in the epi-LASIK corneas (Pvitality was greatest in the epi-LASIK flaps and declined in the LASEK and 100% alcohol flaps (Plive cells. There is also a greater number of proliferating stromal cells following epi-LASIK at 1 week. Based on these in vitro observations, epi-LASIK may result in greater levels of haze compared to LASEK. Copyright 2011, SLACK Incorporated.

Living bacterial sacrificial porogens to engineer decellularized porous scaffolds.

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Feng Xu

Full Text Available Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types.

Rudolf Virchow's medical school dissertation on rheumatism and the cornea: overlooked tribute to the cornea in biomedical research.

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Margo, Curtis E; Harman, Lynn E

2015-02-01

To critique Rudolf Virchow's medical school dissertation on rheumatism and the cornea and to determine whether it might have anticipated his remarkable career in medicine. Review of the English translation of Rudolf Virchow's de Rheumate Praesertim Corneae written in 1843. The dissertation was more than 7000 words long. Virchow considered rheumatism as an irritant disorder not induced by acid as traditionally thought but by albumin. He concluded that inflammation was secondary to a primary irritant and that the "seat" of rheumatism was "gelatinous" (connective) tissues, which included the cornea. He divided kerato-rheumatism into different varieties. The prognosis of keratitis was variable, and would eventually lapse into "scrofulosis, syphilis, or arthritis of the cornea." Virchow's dissertation characterizes rheumatism in terms of chemical and tissue interactions that make little sense in the context of today's knowledge of rheumatic disease and keratitis. Ironically, many of these concepts were made obsolete by the cellular model of disease that Virchow championed. Virchow decided to pursue the study of rheumatism through the cornea because he thought that the cornea was an ideal tissue to study disease. This discernment was passed on to his students whose seminal contributions to general pathology were based on research with the cornea. It is debatable whether Virchow's insight into the importance of the cornea in biomedical research at such an early stage of his career could have predicted his monumental contributions to medicine.

Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering

Science.gov (United States)

Goh, Saik-Kia; Bertera, Suzanne; Olsen, Phillip; Candiello, Joe; Halfter, Willi; Uechi, Guy; Balasubramani, Manimalha; Johnson, Scott; Sicari, Brian; Kollar, Elizabeth; Badylak, Stephen F.; Banerjee, Ipsita

2013-01-01

Approximately 285 million people worldwide suffer from diabetes, with insulin supplementation as the most common treatment measure. Regenerative medicine approaches such as a bioengineered pancreas has been proposed as potential therapeutic alternatives. A bioengineered pancreas will benefit from the development of a bioscaffold that supports and enhances cellular function and tissue development. Perfusion-decellularized organs are a likely candidate for use in such scaffolds since they mimic compositional, architectural and biomechanical nature of a native organ. In this study, we investigate perfusion-decellularization of whole pancreas and the feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our result demonstrates that perfusion-decellularization of whole pancreas effectively removes cellular and nuclear material while retaining intricate three-dimensional microarchitecture with perfusable vasculature and ductal network and crucial extracellular matrix (ECM) components. To mimic pancreatic cell composition, we recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured up to 5 days. Our result shows successful cellular engraftment within the decellularized pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene expression. These findings support biological utility of whole pancreas ECM as a biomaterials scaffold for supporting and enhancing pancreatic cell functionality and represent a step toward bioengineered pancreas using regenerative medicine approaches. PMID:23787110

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

Science.gov (United States)

Goldberg-Bockhorn, Eva; Schwarz, Silke; Subedi, Rachana; Elsässer, Alexander; Riepl, Ricarda; Walther, Paul; Körber, Ludwig; Breiter, Roman; Stock, Karl; Rotter, Nicole

2018-02-01

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO 2 ) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO 2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Favorable Effects of the Detergent and Enzyme Extraction Method for Preparing Decellularized Bovine Pericardium Scaffold for Tissue Engineered Heart Valves

NARCIS (Netherlands)

Yang, Min; Chen, Chang-Zhi; Wang, Xue-Ning; Zhu, Ya-Bin; Gu, Y. John

2009-01-01

Bovine pericardium has been extensively applied as the biomaterial for artificial heart valves and may potentially be used as a scaffold for tissue-engineered heart valves after decellularization. Although various methods of decellularization are currently available, it is unknown which method is

Spherical aberrations of human astigmatic corneas.

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Zhao, Huawei; Dai, Guang-Ming; Chen, Li; Weeber, Henk A; Piers, Patricia A

2011-11-01

To evaluate whether the average spherical aberration of human astigmatic corneas is statistically equivalent to human nonastigmatic corneas. Spherical aberrations of 445 astigmatic corneas prior to laser vision correction were retrospectively investigated to determine Zernike coefficients for central corneal areas 6 mm in diameter using CTView (Sarver and Associates). Data were divided into groups according to cylinder power (0.01 to 0.25 diopters [D], 0.26 to 0.75 D, 0.76 to 1.06 D, 1.07 to 1.53 D, 1.54 to 2.00 D, and >2.00 D) and according to age by decade. Spherical aberrations were correlated with age and astigmatic power among groups and the entire population. Statistical analyses were conducted, and P.05 for all tested groups). Mean spherical aberration of astigmatic corneas was not correlated significantly with cylinder power or age (P>.05). Spherical aberrations are similar to those of nonastigmatic corneas, permitting the use of these additional data in the design of aspheric toric intra-ocular lenses. Copyright 2011, SLACK Incorporated.

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering.

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Neal, Christopher A; Nelson-Brantley, Jennifer G; Detamore, Michael S; Staecker, Hinrich; Mellott, Adam J

2018-01-01

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton's jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

Anatomy and physiology of the cornea.

Science.gov (United States)

DelMonte, Derek W; Kim, Terry

2011-03-01

The importance of the cornea to the ocular structure and visual system is often overlooked because of the cornea's unassuming transparent nature. The cornea lacks the neurobiological sophistication of the retina and the dynamic movement of the lens; yet, without its clarity, the eye would not be able to perform its necessary functions. The complexity of structure and function necessary to maintain such elegant simplicity is the wonder that draws us to one of the most important components of our visual system. Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Modified silk fibroin scaffolds with collagen/decellularized pulp for bone tissue engineering in cleft palate: Morphological structures and biofunctionalities

International Nuclear Information System (INIS)

Sangkert, Supaporn; Meesane, Jirut; Kamonmattayakul, Suttatip; Chai, Wen Lin

2016-01-01

Cleft palate is a congenital malformation that generates a maxillofacial bone defect around the mouth area. The creation of performance scaffolds for bone tissue engineering in cleft palate is an issue that was proposed in this research. Because of its good biocompatibility, high stability, and non-toxicity, silk fibroin was selected as the scaffold of choice in this research. Silk fibroin scaffolds were prepared by freeze-drying before immerging in a solution of collagen, decellularized pulp, and collagen/decellularized pulp. Then, the immersed scaffolds were freeze-dried. Structural organization in solution was observed by Atomic Force Microscope (AFM). The molecular organization of the solutions and crystal structure of the scaffolds were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD), respectively. The weight increase of the modified scaffolds and the pore size were determined. The morphology was observed by a scanning electron microscope (SEM). Mechanical properties were tested. Biofunctionalities were considered by seeding osteoblasts in silk fibroin scaffolds before analysis of the cell proliferation, viability, total protein assay, and histological analysis. The results demonstrated that dendrite structure of the fibrils occurred in those solutions. Molecular organization of the components in solution arranged themselves into an irregular structure. The fibrils were deposited in the pores of the modified silk fibroin scaffolds. The modified scaffolds showed a beta-sheet structure. The morphological structure affected the mechanical properties of the silk fibroin scaffolds with and without modification. Following assessment of the biofunctionalities, the modified silk fibroin scaffolds could induce cell proliferation, viability, and total protein particularly in modified silk fibroin with collagen/decellularized pulp. Furthermore, the histological analysis indicated that the cells could adhere in modified silk fibroin

Customized Finite Element Modelling of the Human Cornea.

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Irene Simonini

Full Text Available To construct patient-specific solid models of human cornea from ocular topographer data, to increase the accuracy of the biomechanical and optical estimate of the changes in refractive power and stress caused by photorefractive keratectomy (PRK.Corneal elevation maps of five human eyes were taken with a rotating Scheimpflug camera combined with a Placido disk before and after refractive surgery. Patient-specific solid models were created and discretized in finite elements to estimate the corneal strain and stress fields in preoperative and postoperative configurations and derive the refractive parameters of the cornea.Patient-specific geometrical models of the cornea allow for the creation of personalized refractive maps at different levels of IOP. Thinned postoperative corneas show a higher stress gradient across the thickness and higher sensitivity of all geometrical and refractive parameters to the fluctuation of the IOP.Patient-specific numerical models of the cornea can provide accurate quantitative information on the refractive properties of the cornea under different levels of IOP and describe the change of the stress state of the cornea due to refractive surgery (PRK. Patient-specific models can be used as indicators of feasibility before performing the surgery.

ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix

DEFF Research Database (Denmark)

Mayorca-Guiliani, Alejandro E.; Madsen, Chris D.; Cox, Thomas R.

2017-01-01

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ...... decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM....... Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over...

Porcine cadaver iris model for iris heating during corneal surgery with a femtosecond laser

Science.gov (United States)

Sun, Hui; Fan, Zhongwei; Wang, Jiang; Yan, Ying; Juhasz, Tibor; Kurtz, Ron

2015-03-01

Multiple femtosecond lasers have now been cleared for use for ophthalmic surgery, including for creation of corneal flaps in LASIK surgery. Preliminary study indicated that during typical surgical use, laser energy may pass beyond the cornea with potential effects on the iris. As a model for laser exposure of the iris during femtosecond corneal surgery, we simulated the temperature rise in porcine cadaver iris during direct illumination by the femtosecond laser. Additionally, ex-vivo iris heating due to femtosecond laser irradiation was measured with an infrared thermal camera (Fluke corp. Everett, WA) as a validation of the simulation.

Serologic Evaluation of Cornea Donors and Microbiologic Evaluation of Cornea Storage Media in an Eye Bank from Izmir, Turkey.

Science.gov (United States)

Palamar, Melis; Degirmenci, Cumali; Sertoz, Ruchan; Aydemir, Sohret; Egrilmez, Sait; Yagci, Ayse

2017-12-01

Our objective was to evaluate the serologic positivity of cornea donors and microbiologic positivity of cornea storage media at the Ege University Tissue and Cornea Bank, Izmir, Turkey. We retrospectively investigated the serologic blood sample and microbiological culture media analysis results of all cornea donors at Ege University Tissue and Cornea Bank between 2007 and 2015 with reference to age, sex, and cause of death of each donor. Mean age of the 955 deceased donors was 43.19 ± 15.89 years (range, 2-65 y). The mean postmortem time to blood sample removal and excision of the cornea tissue was 8.4 hours (range, 4-12 h). Serologic analyses showed that 855 donors (89.5%) were seronegative. The remaining donors were seropositive for hepatitis B (54 donors; 5.7%), human immunodeficiency (27 donors; 2.8%), hepatitis C (14 donors; 1.5%), and syphilis (5 donors; 0.5%) virus infections. Microbiologic analyses of the storage media were negative, with no microorganisms shown in 855 samples (89.5%). Candida species (32 donors; 3.4%), Escherichia coli (14 donors; 1.5%), Pseudomonas aeruginosa (11 donors; 1.2%), methicillin-resistant Staphylococcus aureus (11 donors; 1.2%), Enterobacter species (11 donors; 1.2%), Klebsiella pneumoniae (7 donors; 0.7%), Acinetobacter baumannii (6 donors; 0.6%), Proteus species (5 donors; 0.5%), and Corynebacterium species (3 donors; 0.3%) were the detected microorganisms in the infected storage media. False-positive serologic results among cornea donors were high. The incidence of false-positive results might be decreased by earlier blood removal from deceased donors and testing of all potential donors in intensive care units. Although rare, endophthalmitis after keratoplasty might be a devastating problem. In addition to serologic testing, microbiologic analyses of cornea storage media before transplant may be an effective way to prevent postoperative infectious complications.

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

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Yalong Dang

2017-08-01

Full Text Available Objective The trabecular meshwork (TM is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F ablation (−80 °C × 2, to 0.02% saponin (S treatment, or the control group (C, respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64. IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed

Tissue engineering by decellularization and 3D bioprinting

OpenAIRE

Garreta, Elena; Oria, Roger; Tarantino, Carolina; Pla Roca, Mateu; Prado, Patricia; Fernández Avilés, Francisco; Campistol Plana, Josep M.; Samitier i Martí, Josep; Montserrat, Núria

2017-01-01

Discarded human donor organs have been shown to provide decellularized extracellular matrix (dECM) scaffolds suitable for organ engineering. The quest for appropriate cell sources to satisfy the need of multiple cells types in order to fully repopulate human organ-derived dECM scaffolds has opened new venues for the use of human pluripotent stem cells (hPSCs) for recellularization. In addition, three-dimensional (3D) bioprinting techniques are advancing towards the fabrication of biomimetic c...

Facts about the Cornea and Corneal Disease

Science.gov (United States)

... most used keratoprosthesis. It is made of clear plastic and consists of three parts, with donor cornea ... that, if successful, will lead to a significant reduction in the transmission of HSV to the cornea. ...

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

Science.gov (United States)

Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix liver scaffolds.

Science.gov (United States)

Sabetkish, Shabnam; Kajbafzadeh, Abdol-Mohammad; Sabetkish, Nastaran; Khorramirouz, Reza; Akbarzadeh, Aram; Seyedian, Sanam Ladi; Pasalar, Parvin; Orangian, Saghar; Beigi, Reza Seyyed Hossein; Aryan, Zahra; Akbari, Hesam; Tavangar, Seyyed Mohammad

2015-04-01

To report the results of whole liver decellularization by two different methods. To present the results of grafting rat and sheep decellularized liver matrix (DLM) into the normal rat liver and compare natural cell seeding process in homo/xenograft of DLM. To compare the results of in vitro whole liver recellularization with rats' neonatal green fluorescent protein (GFP)-positive hepatic cells with outcomes of in vivo recellularization process. Whole liver of 8 rats and 4 sheep were resected and cannulated via the hepatic vein and perfused with sodium dodecyl sulfate (SDS) or Triton + SDS. Several examinations were performed to compare the efficacy of these two decellularization procedures. In vivo recellularization of sheep and rat DLMs was performed following transplantation of multiple pieces of both scaffolds in the subhepatic area of four rats. To compare the efficacy of different scaffolds in autologous cell seeding, biopsies of homograft and xenograft were assessed 8 weeks postoperatively. Whole DLMs of 4 rats were also recellularized in vitro by perfusion of rat's fetal GFP-positive hepatic cells with pulsatile bioreactor. Histological evaluation and enzymatic assay were performed for both in vivo and in vitro recellularized samples. The results of this study demonstrated that the triton method was a promising decellularization approach for preserving the three-dimensional structure of liver. In vitro recellularized DLMs were more similar to natural ones compared with in vivo recellularized livers. However, homografts showed better characteristics with more organized structure compared with xenografts. In vitro recellularization of liver scaffolds with autologous cells represents an attractive prospective for regeneration of liver as one of the most compound organs. In vivo cell seeding on the scaffold of the same species may have more satisfactory outcomes when compared with the results of xenotransplantation. This study theoretically may pave the road for

Dynamic Sensing of Cornea Deformation during an Air Puff

Science.gov (United States)

Yamada, Kenji; Yamasaki, Naoyuki; Gosho, Takumi; Kiuchi, Yoshiaki; Takenaka, Jouji; Higashimori, Mitsuru; Kaneko, Makoto

In early diagnosis of glancoma, intraocular pressure measurement is one of an important method. Non-contact method has measured eye pressure through the deformation of cornea during the increase of the force due to air puff. The deformation is influenced by the cornea stiffness as well as the eye internal pressure. Since the cornea stiffness is unknown in general, it is difficult to evaluate the ture eye pressure. The dynamic behavior of cornea under air puff may provide us with a good hint for evaluating the cornea stiffness appropriately. For this purpose, we develop the sensing system composed of a high speed camera, a mirror for producing a virtual camera, a non-contact tonometer and a slit light source. This system enables us to measure the cornea deformation under concave shape. We show the experimental data for human eyes as well as an artificial eye made by transparent material.

Finite element modelling of cornea mechanics: a review

Directory of Open Access Journals (Sweden)

Talisa Mohammad Nejad

2014-01-01

Full Text Available The cornea is a transparent tissue in front of the eye that refracts light and facilitates vision. A slight change in the geometry of the cornea remarkably affects the optical power. Because of this sensitivity, biomechanical study of the cornea can reveal much about its performance and function. In vivo and in vitro studies have been conducted to investigate the mechanics of the cornea and determine its characteristics. Numerical techniques such as the finite element method (FEM have been extensively implemented as effective and noninvasive methods for analyzing corneal mechanics and possible disorders. This article reviews the use of FEM for assessing the mechanical behavior of the cornea. Different applications of FEM in corneal disease studies, surgical predictions, impact simulations, and clinical applications have been reviewed. Some suggestions for the future of this type of modeling in the area of corneal mechanics are also discussed.

Tissue engineering approaches to develop decellularized tendon matrices functionalized with progenitor cells cultured under undifferentiated and tenogenic conditions

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Daniele D’Arrigo

2017-11-01

Full Text Available Tendon ruptures and retractions with an extensive tissue loss represent a major clinical problem and a great challenge in surgical reconstruction. Traditional approaches consist in autologous or allogeneic grafts, which still have some drawbacks. Hence, tissue engineering strategies aimed at developing functionalized tendon grafts. In this context, the use of xenogeneic tissues represents a promising perspective to obtain decellularized tendon grafts. This study is focused on the identification of suitable culture conditions for the generation of reseeded and functional decellularized constructs to be used as tendon grafts. Equine superficial digital flexor tendons were decellularized, reseeded with mesenchymal stem cells (MSCs from bone marrow and statically cultured in two different culture media to maintain undifferentiated cells (U-MSCs or to induce a terminal tenogenic differentiation (T-MSCs for 24 hours, 7 and 14 days. Cell viability, proliferation, morphology as well as matrix deposition and type I and III collagen production were assessed by means of histological, immunohistochemical and semi-quantitative analyses. Results showed that cell viability was not affected by any culture conditions and active proliferation was maintained 14 days after reseeding. However, seeded MSCs were not able to penetrate within the dense matrix of the decellularized tendons. Nevertheless, U-MSCs synthesized a greater amount of extracellular matrix rich in type I collagen compared to T-MSCs. In spite of the inability to deeply colonize the decellularized matrix in vitro, reseeding tendon matrices with U-MSCs could represent a suitable method for the functionalization of biological constructs, considering also any potential chemoattractant capability of the newly deposed extracellular matrix to recruit resident cells. This bioengineering approach can be exploited to produce functionalized tendon constructs for the substitution of large tendon defects.

Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

KAUST Repository

Gerhardt, Lutz Christian; Widdows, Kate L.; Erol, Melek M.; Nandakumar, Anandkumar; Roqan, Iman S.; Ansari, Tahera I.; Boccaccini, Aldo R.

2012-01-01

amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm 2), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating

[The 2009 performance report of the German cornea banks].

Science.gov (United States)

Schrage, N; Reinhard, T; Seitz, B; Hermel, M; Böhringer, D; Reinshagen, H

2011-03-01

In Germany, human tissue for corneal and amniotic transplantation is supplied by 27 cornea banks. The Section for Tissue Transplantation and Biotechnology of the German Ophthalmological Society records the cornea banks' activities by means of an annual questionnaire. In 2009, a total of 4,818 corneal grafts were processed by 21 responding cornea banks, and 57% were deemed suitable for transplantation. This ratio is slightly higher than the European average. In addition, German cornea banks released 1,257 amniotic grafts in 2009. German cornea banks are currently facing new regulatory issues due to updated legislation regarding tissue transplantation. Recent updates in European law have limited the cutoff time for postmortem blood sampling to 24 h, and this regulation may lead to a significant reduction in potential donors.

Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.

Science.gov (United States)

Caralt, M; Uzarski, J S; Iacob, S; Obergfell, K P; Berg, N; Bijonowski, B M; Kiefer, K M; Ward, H H; Wandinger-Ness, A; Miller, W M; Zhang, Z J; Abecassis, M M; Wertheim, J A

2015-01-01

The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Feasibility of pig and human-derived aortic valve interstitial cells seeding on fixative-free decellularized animal pericardium.

Science.gov (United States)

Santoro, Rosaria; Consolo, Filippo; Spiccia, Marco; Piola, Marco; Kassem, Samer; Prandi, Francesca; Vinci, Maria Cristina; Forti, Elisa; Polvani, Gianluca; Fiore, Gianfranco Beniamino; Soncini, Monica; Pesce, Maurizio

2016-02-01

Glutaraldehyde-fixed pericardium of animal origin is the elective material for the fabrication of bio-prosthetic valves for surgical replacement of insufficient/stenotic cardiac valves. However, the pericardial tissue employed to this aim undergoes severe calcification due to chronic inflammation resulting from a non-complete immunological compatibility of the animal-derived pericardial tissue resulting from failure to remove animal-derived xeno-antigens. In the mid/long-term, this leads to structural deterioration, mechanical failure, and prosthesis leaflets rupture, with consequent need for re-intervention. In the search for novel procedures to maximize biological compatibility of the pericardial tissue into immunocompetent background, we have recently devised a procedure to decellularize the human pericardium as an alternative to fixation with aldehydes. In the present contribution, we used this procedure to derive sheets of decellularized pig pericardium. The decellularized tissue was first tested for the presence of 1,3 α-galactose (αGal), one of the main xenoantigens involved in prosthetic valve rejection, as well as for mechanical tensile behavior and distensibility, and finally seeded with pig- and human-derived aortic valve interstitial cells. We demonstrate that the decellularization procedure removed the αGAL antigen, maintained the mechanical characteristics of the native pig pericardium, and ensured an efficient surface colonization of the tissue by animal- and human-derived aortic valve interstitial cells. This establishes, for the first time, the feasibility of fixative-free pericardial tissue seeding with valve competent cells for derivation of tissue engineered heart valve leaflets. © 2015 Wiley Periodicals, Inc.

Factors influencing the virological testing of cornea donors.

Science.gov (United States)

Röck, Tobias; Beck, Robert; Jürgens, Stefan; Bartz-Schmidt, Karl Ulrich; Bramkamp, Matthias; Thaler, Sebastian; Röck, Daniel

2017-11-01

To assess the influence of donor, environment, and logistical factors on the results of virological testing of blood samples from cornea donors.Data from 670 consecutive cornea donors were analyzed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the results of virological testing of blood samples from cornea donors.The mean annual rate of donors with serology-reactive or not evaluable result was 14.8% (99 of 670) (range 11.9%-16.9%). The cause of donor death by cancer increased the risk of serology-reactive or not evaluable result (P = .0300). Prolonged time between death and post mortem blood removal was associated with a higher rate of serology-reactive or not evaluable result (P donors, sex, and donor age had no significant impact on the results of virological testing of blood samples from cornea donors.The cause of donor death by cancer and a prolonged time between death and post mortem blood removal seem to be mainly responsible for serology-reactive or not evaluable result of blood samples from cornea donors. The percentage of discarded corneas caused by serology-reactive or not evaluable result may be reduced by shortening the period of time between death and post mortem blood removal. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

A Comparative Study of Rat Lung Decellularization by Chemical Detergents for Lung Tissue Engineering

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Hamid Tebyanian

2017-12-01

CONCLUSION: Decellularized lung tissue can be used in the laboratory to study various aspects of pulmonary biology and physiology and also, these results can be used in the continued improvement of engineered lung tissue.

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

Science.gov (United States)

Tang, Zhongshu; Zhang, Fan; Li, Yang; Arjunan, Pachiappan; Kumar, Anil; Lee, Chunsik; Li, Xuri

2011-01-01

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea. PMID:21876523

Decellularized Swine Dental Pulp as a Bioscaffold for Pulp Regeneration

OpenAIRE

Hu, Lei; Gao, Zhenhua; Xu, Junji; Zhu, Zhao; Fan, Zhipeng; Zhang, Chunmei; Wang, Jinsong; Wang, Songlin

2017-01-01

Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM) is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental p...

Mass Spectrometric Analyses of Phosphatidylcholines in Alkali-Exposed Corneal Tissue

Science.gov (United States)

Crane, Ashley M.; Hua, Hong-Uyen; Coggin, Andrew D.; Gugiu, Bogdan G.; Lam, Byron L.; Bhattacharya, Sanjoy K.

2012-01-01

Purpose. The aims were to determine whether exposure to sodium hydroxide results in predictable changes in phosphatidylcholine (PC) in corneal tissue and if PC profile changes correlate to exposure duration. PCs are major components of the cell membrane lipid bilayer and are often involved in biological processes such as signaling. Methods. Enucleated porcine (n = 140) and cadaver human eyes (n = 20) were exposed to water (control) and 11 M NaOH. The corneas were excised and lipids were extracted using the Bligh and Dyer method with suitable modifications. Class-specific lipid identification was carried out using a ratiometric lipid standard on a TSQ Quantum Access Max mass spectrometer. Protein amounts were determined using Bradford assays. Results. Control and alkali-treated corneas showed reproducible PC spectra for both porcine and human corneas. Over 200 PCs were identified for human and porcine control and each experimental time point. Several PC species (m/z values) consequent upon alkali exposure could not be ascribed to a recorded PC species. Control and treated groups showed 41 and 29 common species among them for porcine and human corneas, respectively. The unique PC species peaked at 12 minutes and at 30 minutes for human and porcine corneas followed by a decline consistent with an interplay of alkali penetration and hydrolyses at various time points. Conclusions. Alkali exposure dramatically changes the PC profile of cornea. Our data are consistent with penetration and hydrolysis as stochastic contributors to changes in PCs due to exposure to alkali for a finite duration and amount. PMID:22956606

GENDER AND AGE RELATED VARIATION IN CORNEA POWER

African Journals Online (AJOL)

2014-05-01

May 1, 2014 ... population and how it varies with gender and age. Design: A .... horizontal in orientation. For the purpose of .... race as cornea curvature have been noted to exhibit ... Grosevenor, T. Role of Cornea in Emmetropia and. Myopia.

A bilaminated decellularized scaffold for islet transplantation: Structure, properties and functions in diabetic mice.

Science.gov (United States)

Wang, Xi; Wang, Kai; Zhang, Wei; Qiang, Ming; Luo, Ying

2017-09-01

Ectopic transplantation of islets provides a beta cell-replacement approach that may allow the recovery of physiological regulation of the blood sugar level in patients with Type I diabetes (T1D). In development of new extrahepatic islet transplantation protocols in support of the islet engraftment, it is pivotal to develop scaffold materials with multifaceted functions to provide beneficial microenvironment, mediate host response in favor of vascularization/islet integration and maintain long-term islet function at the transplantation site. In this study, a new composite bilaminar decellularized scaffold (CDS) was fabricated with differential structural, degradation and mechanical properties by the combination of a fast-degrading porous collagen matrix and a mechanically supportive porcine pericardium. When investigated in the epididymal fat pad in syngeneic mouse models, it was shown that CDS could serve as superior scaffolds to promote islet adhesion and viability, and islet-CDS constructs also allowed rapid reversal of the hyperglycemic condition in the host. The engraftment and effects of islets were achieved at low islet numbers, accompanied by minimal adverse tissue reactions and optimal islet integration with the surrounding fat tissue. The bioactive surface, mechanical/chemical durability and biocompatibility of the CDS may all have played important roles in facilitating the engraftment of islets. Our study provided new insights into scaffold's function in the interplay of cells, materials and host tissue and the extracellular matrix-based scaffolds have potential for clinical translation in the beta cell-replacement therapy to treat T1D. Copyright © 2017 Elsevier Ltd. All rights reserved.

ANALYSIS OF DONOR CORNEA RETRIEVAL DATA- HOSPITAL AND RESIDENCES

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Rekha Gyanchand

2017-07-01

Full Text Available BACKGROUND The voluntary eye donation is still the most popular methods of eye donation in our country. Since last three decades, the voluntary eye donation is promoted by a number of NGOs and Government of India. The meaning of voluntary eye donation is that an individual has prior knowledge about eye donation and also has the contact number of the eye bank. When they come across any death in their community, they will contact the eye bank for eye donation. As per Eye Bank Association of India, the incidence of total blindness is 8.9 million and 1% of this is corneal blindness. Each year, 25,000 to 30,000/new corneal blind patients are added. The voluntary eye donation is not sufficient in providing transplantable corneas to the corneal blind. As our concentration is mainly on voluntary eye collections, this is one of the main reasons that we are unable reach our target of sight restoration in the management of corneal blindness. In this study, we have analysed the voluntary eye donations procured from residence and hospital. The aim of the study is to compare corneas procured by hospital eye donations and residence eye donations to look into all factors that contribute to a transplantable cornea. MATERIALS AND METHODS The donor corneas retrieved from the year 2005-2010 were analysed. The hospital eye donations and residential eye donations were divided into two study groups. The factors analysed were number of corneas collected, donor age and gender, serology test result, death to preservation time (DPT, optical grade cornea, utilisation of corneas for transplantation and role of pledging in eye donation. Study Type- Retrospective study. Study Done- At Lions International Eye Bank, Bangalore. RESULTS The total number of donor eyes collected from the year 2005-2010 were 7362 of which residence collections was 50.73% and hospital collections was 49.26%. The 5-year period between from 2005 to 2010 in spite of not conducting a Hospital Cornea

Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications.

Directory of Open Access Journals (Sweden)

Daniel W Youngstrom

Full Text Available Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS, trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.

Repopulating Decellularized Kidney Scaffolds: An Avenue for Ex Vivo Organ Generation

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Robert A. McKee

2016-03-01

Full Text Available Recent research has shown that fully developed organs can be decellularized, resulting in a complex scaffold and extracellular matrix (ECM network capable of being populated with other cells. This work has resulted in a growing field in bioengineering focused on the isolation, characterization, and modification of organ derived acellular scaffolds and their potential to sustain and interact with new cell populations, a process termed reseeding. In this review, we cover contemporary advancements in the bioengineering of kidney scaffolds including novel work showing that reseeded donor scaffolds can be transplanted and can function in recipients using animal models. Several major areas of the field are taken into consideration, including the decellularization process, characterization of acellular and reseeded scaffolds, culture conditions, and cell sources. Finally, we discuss future avenues based on the advent of 3D bioprinting and recent developments in kidney organoid cultures as well as animal models of renal genesis. The ongoing mergers and collaborations between these fields hold the potential to produce functional kidneys that can be generated ex vivo and utilized for kidney transplantations in patients suffering with renal disease.

Matrix metalloproteinase expression in excimer laser wounded rabbit corneas

Science.gov (United States)

Hahn, Taewon; Chamon, Wallace; Akova, Yonja; Stark, Walter J.; Stetler-Stevenson, William G.; Azar, Dimitri T.

1994-06-01

This study was performed to obtain information about matrix metalloproteinase (MMP) expression in excimer-wounded corneas and to determine whether MMPs expression correlates with the depth of the ablation. 6-mm excimer keratectomy (60 or 180 micrometers ) was performed using the 193-mm ArF excimer laser on 12 NZW rabbits. Corneas treated with mechanical epithelial debridement and untreated corneas served as controls. Rabbits were killed at 20 and 30 hr after laser ablation. Zymography after SDS extraction was performed on regenerated central epithelium and the central stroma to determine MMPs expression. We observed enzymatic activity of a 92 KDa band in the epithelium of excimer-ablated corneas but not in that following debridement wounds and untreated controls. The expression of the 92 KDa MMP was most pronounced with the deeper excimer ablation. A 72 KDa band of enzymatic activity present in the stroma of all treated and control eyes was also seen in the epithelium of excimer-ablated corneas. These proteolytic enzymes may play an important role in wound healing and remodelling after excimer keratectomy.

File list: ALL.Oth.50.AllAg.Cornea [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Oth.50.AllAg.Cornea mm9 All antigens Others Cornea SRX437637,SRX437636,SRX24830...2,SRX248301,SRX437638 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Oth.50.AllAg.Cornea.bed ...

File list: ALL.Oth.05.AllAg.Cornea [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Oth.05.AllAg.Cornea mm9 All antigens Others Cornea SRX437637,SRX437636,SRX24830...2,SRX248301,SRX437638 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Oth.05.AllAg.Cornea.bed ...

File list: ALL.Oth.20.AllAg.Cornea [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Oth.20.AllAg.Cornea mm9 All antigens Others Cornea SRX437637,SRX437636,SRX24830...2,SRX248301,SRX437638 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Oth.20.AllAg.Cornea.bed ...

File list: ALL.Oth.10.AllAg.Cornea [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Oth.10.AllAg.Cornea mm9 All antigens Others Cornea SRX437637,SRX248302,SRX43763...6,SRX248301,SRX437638 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Oth.10.AllAg.Cornea.bed ...

Extracellular matrix alterations in human corneas with bullous keratopathy

DEFF Research Database (Denmark)

Ljubimov, A V; Burgeson, R E; Butkowski, R J

1996-01-01

PURPOSE. To uncover abnormalities of extracellular matrix (ECM) distribution in human corneas with pseudophakic and aphakic bullous keratopathy (PBK/ABK). METHODS. Indirect immunofluorescence with antibodies to 27 ECM components was used on frozen sections of 14 normal and 20 PBK/ABK corneas...... in some cases, correlated with decreased visual acuity. In normal central corneas, tenascin was never found. Other major ECM abnormalities in PBK/ABK corneas compared to normals included: discontinuous epithelial BM straining for laminin-1 (alpha 1 beta 1 gamma 1), entactin/nidogen and fibronectin......; accumulation of fibronectin and alpha 1-alpha 2 type IV collagen on the endothelial face of the Descemet's membrane; and abnormal deposition of stromal ECM (tenascin, fibronectin, decorin, types I, III, V, VI, VIII, XII, XIV collagen) and BM components (type IV, collagen, perlecan, bamacan, laminin-1, entactin...

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials.

Science.gov (United States)

Wang, Raymond M; Johnson, Todd D; He, Jingjin; Rong, Zhili; Wong, Michelle; Nigam, Vishal; Behfar, Atta; Xu, Yang; Christman, Karen L

2017-06-01

Current assessment of biomaterial biocompatibility is typically implemented in wild type rodent models. Unfortunately, different characteristics of the immune systems in rodents versus humans limit the capability of these models to mimic the human immune response to naturally derived biomaterials. Here we investigated the utility of humanized mice as an improved model for testing naturally derived biomaterials. Two injectable hydrogels derived from decellularized porcine or human cadaveric myocardium were compared. Three days and one week after subcutaneous injection, the hydrogels were analyzed for early and mid-phase immune responses, respectively. Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials. The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4 + T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice. T-helper cells, in response to the allogeneic hydrogel material, were also less polarized towards a pro-remodeling Th2 phenotype compared to xenogeneic extracellular matrix hydrogels in humanized mice. In both models, both biomaterials induced the infiltration of macrophages polarized towards a M2 phenotype and T-helper cells polarized towards a Th2 phenotype. In conclusion, these studies showed the importance of testing naturally derived biomaterials in immune competent animals and the potential of utilizing this humanized mouse model for further studying human immune cell responses to biomaterials in an in vivo environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures.

Directory of Open Access Journals (Sweden)

Chiara Gardin

Full Text Available The combination of bone grafting materials with guided bone regeneration (GBR membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM. In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications.

Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

International Nuclear Information System (INIS)

Liu, Xiaozhen; Zhou, Long; Chen, Xi; Liu, Tao; Pan, Guoqing; Cui, Wenguo; Li, Mao; Luo, Zong-Ping; Pei, Ming; Yang, Huilin; Gong, Yihong; He, Fan

2016-01-01

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H_2O_2) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100 μM H_2O_2 dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs. - Highlights: • Decellularization preserved the architecture and components of cell-deposited ECM. �

Culturing on decellularized extracellular matrix enhances antioxidant properties of human umbilical cord-derived mesenchymal stem cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Xiaozhen [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); Zhou, Long; Chen, Xi [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Liu, Tao [Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pan, Guoqing; Cui, Wenguo; Li, Mao; Luo, Zong-Ping [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Pei, Ming [Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV 26506 (United States); Yang, Huilin [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China); Gong, Yihong, E-mail: gongyih@mail.sysu.edu.cn [School of Engineering, Sun Yat-sen University, Guangzhou 510006 (China); He, Fan, E-mail: fanhe@suda.edu.cn [Orthopaedic Institute, Soochow University, Suzhou 215007 (China); Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou 215006 (China)

2016-04-01

Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have attracted great interest in clinical application because of their regenerative potential and their lack of ethical issues. Our previous studies showed that decellularized cell-deposited extracellular matrix (ECM) provided an in vivo-mimicking microenvironment for MSCs and facilitated in vitro cell expansion. This study was conducted to analyze the cellular response of UC-MSCs when culturing on the ECM, including reactive oxygen species (ROS), intracellular antioxidative enzymes, and the resistance to exogenous oxidative stress. After decellularization, the architecture of cell-deposited ECM was characterized as nanofibrous, collagen fibrils and the matrix components were identified as type I and III collagens, fibronectin, and laminin. Compared to tissue culture polystyrene (TCPS) plates, culturing on ECM yielded a 2-fold increase of UC-MSC proliferation and improved the percentage of cells in the S phase by 2.4-fold. The levels of intracellular ROS and hydrogen peroxide (H{sub 2}O{sub 2}) in ECM-cultured cells were reduced by 41.7% and 82.9%, respectively. More importantly, ECM-cultured UC-MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase, up-regulated expression of silent information regulator type 1, and suppressed phosphorylation of p38 mitogen-activated protein kinase. Furthermore, a continuous treatment with exogenous 100 μM H{sub 2}O{sub 2} dramatically inhibited osteogenic differentiation of UC-MSCs cultured on TCPS, but culturing on ECM retained the differentiation capacity for matrix mineralization and osteoblast-specific marker gene expression. Collectively, by providing sufficient cell amounts and enhancing antioxidant capacity, decellularized ECM can be a promising cell culture platform for in vitro expansion of UC-MSCs. - Highlights: • Decellularization preserved the architecture and components of cell

Corneal-protective effects of an artificial tear containing sodium hyaluronate and castor oil on a porcine short-term dry eye model.

Science.gov (United States)

Hasegawa, Takashi; Amako, Hideki; Yamamoto, Takeshi; Tazawa, Mariko; Sakamoto, Yuji

2014-09-01

The corneal-protective effects of an artificial tear containing sodium hyaluronate (SH) and castor oil (CO) were evaluated on a porcine short-term dry eye model. Fresh porcine eyes with an intact cornea were treated with an artificial tear of saline, SH solution (0.1%, 0.5% or 1%), CO solution (0.5%, 1% or 5%) or a mixture solution containing 0.5% SH and 1% CO and then desiccated for 60, 90 or 180 min. To assess corneal damage, the eyes were stained with methylene blue (MB) or lissamine green (LG). The staining score of MB, absorbance of MB extracted from the cornea and staining density of LG increased significantly with increasing desiccation time in untreated and all artificial tear-treated eyes, although there were no significant differences in staining scores and absorbance of MB between eyes treated continuously with saline and 1% SH-treated ones at 60 and 90 min of desiccation or the mixture-treated eyes at 60 min of desiccation. No significant differences in the staining density of LG were also found between continuous saline-treated eyes and ones desiccated for 60 min and treated with 1% SH and the mixture. Mild cytoplasmic vacuolations were histopathologically observed in the basal and wing cells in eyes desiccated for 60 min and treated with 1% SH and the mixture. The mixture solution containing 0.5% SH and 1% CO has protective effects against corneal desiccation similar to those of 1% SH and would be helpful as an artificial tear.

Pharmacokinetics of chlorhexidine gluconate 0.02% in the rabbit cornea.

Science.gov (United States)

Xuguang, Sun; Yanchuang, Liang; Feng, Zhang; Shiyun, Luo; Xiaotang, Yin

2006-08-01

The aim of this study was to determine the pharmacokinetic parameters of chlorhexidine gluconate (CHG) in the rabbit cornea. Each eye of 16 New Zealand white rabbits were topically instilled with 50 microL of CHG 0.02% eye drops twice with a 5-min interval. Four (4) corneas of 2 rabbits were harvested at each time point. The concentration of CHG in the cornea was determined with high-performance liquid chromatography (HPLC) and 387 software to simulate the pharmacokinetic parameters. The concentration of CHG in the cornea displayed an open two-compartment model. Tmax was 13.75 min, Cmax 0.713 microg.g1, clearance rate 1.64 microg.g-1.min-1, and t1/2alpha, t1/2beta, and t1/2ka was 2.65, 48.72, and 2.67 min, respectively. The concentration of CHG in the rabbit cornea could be determined by means of HPLC. The maximum concentration of CHG in the corneal tissue was much higher than the trophozoite minimum amoebicidal concentration (TMAC) in vitro.

CORNEA AND ANTERIOR SEGMENT

African Journals Online (AJOL)

Yet, fewer than 150,000 corneal transplant surgeries are performed annually worldwide. Corneal blindness arises from a wide variety of causes, from mechanical, chemical, and radiation injury to microbial infections and corneal degenerative diseases. The use of human cadaver donor corneas has been limited by its often.

Melatonin Entrains PER2::LUC Bioluminescence Circadian Rhythm in the Mouse Cornea

Science.gov (United States)

Baba, Kenkichi; Davidson, Alec J.; Tosini, Gianluca

2015-01-01

Purpose Previous studies have reported the presence of a circadian rhythm in PERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in mouse photoreceptors, retina, RPE, and cornea. Melatonin (MLT) modulates many physiological functions in the eye and it is believed to be one of the key circadian signals within the eye. The aim of the present study was to investigate the regulation of the PER2::LUC circadian rhythm in mouse cornea and to determine the role played by MLT. Methods Corneas were obtained from PER2::LUC mice and cultured to measure bioluminescence rhythmicity in isolated tissue using a Lumicycle or CCD camera. To determine the time-dependent resetting of the corneal circadian clocks in response to MLT or IIK7 (a melatonin type 2 receptor, MT2, agonist) was added to the cultured corneas at different times of the day. We also defined the location of the MT2 receptor within different corneal layers using immunohistochemistry. Results A long-lasting bioluminescence rhythm was recorded from cultured PER2::LUC cornea and PER2::LUC signal was localized to the corneal epithelium and endothelium. MLT administration in the early night delayed the cornea rhythm, whereas administration of MLT at late night to early morning advanced the cornea rhythm. Treatment with IIK7 mimicked the MLT phase-shifting effect. Consistent with these results, MT2 immunoreactivity was localized to the corneal epithelium and endothelium. Conclusions Our work demonstrates that MLT entrains the PER2::LUC bioluminescence rhythm in the cornea. Our data indicate that the cornea may represent a model to study the molecular mechanisms by which MLT affects the circadian clock. PMID:26207312

Induction of autoimmune abdominal aortic aneurysm in pigs

DEFF Research Database (Denmark)

Riber, Sara Schødt; Ali, Mulham; Bergseth, Sara Hveding

2017-01-01

of this study was to develop a large animal model for abdominal aortic aneurysm induction through autoimmunity by performing sheep-to-pig xenotransplantation. Methods Six pigs underwent a xenotransplantation procedure where the infrarenal porcine aorta was replaced by a decellularized sheep aorta...

Advanced Squamous Cell Carcinoma of Cornea in a Child with ...

African Journals Online (AJOL)

lids, conjunctiva, and cornea are affected by the disease process.[3] A case of XP ... In the left eye, almost the entire cornea was involved with a grayish-white, irregular, and ... referring to its characteristic dry pigmented skin. It was also named ...

Correlation between epithelial thickness in normal corneas, untreated ectatic corneas, and ectatic corneas previously treated with CXL; is overall epithelial thickness a very early ectasia prognostic factor?

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Kanellopoulos AJ

2012-05-01

Full Text Available Anastasios John Kanellopoulos,1,2 Ioannis M Aslanides,3 George Asimellis11Laservision Eye Institute, Athens, 2Emmetropia Mediterranean Eye Clinic, Crete, Greece, 3New York University School of Medicine, NY, USAPurpose: To determine and correlate epithelial corneal thickness (pachymetric measurements taken with a digital arc scanning very high frequency ultrasound biomicroscopy (HF UBM imaging system (Artemis-II, and compare mean and central epithelial thickness among normal eyes, untreated keratoconic eyes, and keratoconic eyes previously treated with collagen crosslinking (CXL.Methods: Epithelial pachymetry measurements (topographic mapping were conducted on 100 subjects via HF UBM. Three groups of patients were included: patients with normal eyes (controls, patients with untreated keratoconic eyes, and patients with keratoconic eyes treated with CXL. Central, mean, and peripheral corneal epithelial thickness was examined for each group, and a statistical study was conducted.Results: Mean, central, and peripheral corneal epithelial thickness was compared between the three groups of patients. Epithelium thickness varied substantially in the keratoconic group, and in some cases there was a difference of up to 20 µm between various points of the same eye, and often a thinner epithelium coincided with a thinner cornea. However, on average, data from the keratoconic group suggested an overall thickening of the epithelium, particularly over the pupil center of the order of +3 µm, while the mean epithelium thickness was on average +1.1 µm, compared to the control population (P = 0.005. This overall thickening was more pronounced in younger patients in the keratoconic group. Keratoconic eyes previously treated with CXL showed, on average, virtually the same average epithelium thickness (mean –0.7 µm, –0.2 µm over the pupil center, –0.9 µm over the peripheral zone as the control group. This finding further reinforces our novel theory of the

Analysis of Riboflavin Compounds in the Rabbit Cornea In Vivo.

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Hammer, Arthur; Rudaz, Serge; Guinchard, Sylvie; Kling, Sabine; Richoz, Olivier; Hafezi, Farhad

2016-09-01

To investigate the composition and concentration of individual riboflavin compounds in the corneal stroma in vivo after soaking with various commercially available riboflavin formulations. Experiments were performed in 26 rabbit corneas in vivo: 24 corneas were soaked with riboflavin formulations for 30 minutes or with 0.9% NaCl for control (n = 2). After treatment, corneas were excised and prepared for ultra-high-pressure liquid chromatography (UHPLC) analysis. Additionally, computational chemical analysis of riboflavin compounds and keratan sulfate were performed. The amount of riboflavin and riboflavin phosphate isomers in cornea decreased by a factor of 10 to 100, when compared to the amount in riboflavin formulations. In particular, we found an inverse relationship in the ratio of riboflavin to riboflavin phosphate isomer concentration between formulations and cornea. The electronegativity and ionization potential of riboflavin and phosphate isomers are different. The inverse relationship observed might be explained by a stronger electronegativity of the phosphate isomers, leading to a stronger repulsion by corneal proteoglycans. Indicating the individual concentration of riboflavin compounds in formulations is more representative than the total riboflavin concentration. Riboflavin formulations and CXL protocols might be improved considering the differences in diffusion and ionization potentials of the different riboflavin compounds.

Clinical and epidemiological aspects of cornea transplant patients of a reference hospital 1

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Cruz, Giovanna Karinny Pereira; de Azevedo, Isabelle Campos; Carvalho, Diana Paula de Souza Rego Pinto; Vitor, Allyne Fortes; Santos, Viviane Euzébia Pereira; Ferreira, Marcos Antonio

2017-01-01

ABSTRACT Objective: clinically characterizing cornea transplant patients and their distribution according to indicated and post-operative conditions of cornea transplantation, as well as estimating the average waiting time. Method: a cross-sectional, descriptive and analytical study performed for all cornea transplants performed at a reference service (n=258). Data were analyzed using Statistical Package for the Social Sciences, version 20.0. Results: the main indicator for cornea transplant was keratoconus. The mean waiting time for the transplant was approximately 5 months and 3 weeks for elective transplants and 9 days for urgent cases. An association between the type of corneal disorder with gender, age, previous surgery, eye classification, glaucoma and anterior graft failure were found. Conclusion: keratoconus was the main indicator for cornea transplant. Factors such as age, previous corneal graft failure (retransplantation), glaucoma, cases of surgeries prior to cornea transplant (especially cataract surgery) may be related to the onset corneal endothelium disorders. PMID:28614429

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

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Bukowiecki, Anne; Hos, Deniz; Cursiefen, Claus; Eming, Sabine A

2017-06-12

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.

Assessment of UVA-Riboflavin Corneal Cross-Linking Using Small Amplitude Oscillatory Shear Measurements.

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Aslanides, Ioannis M; Dessi, Claudia; Georgoudis, Panagiotis; Charalambidis, Georgios; Vlassopoulos, Dimitris; Coutsolelos, Athanassios G; Kymionis, George; Mukherjee, Achyut; Kitsopoulos, Theofanis N

2016-04-01

The effect of ultraviolet (UV)-riboflavin cross-linking (CXL) has been measured primarily using the strip extensometry technique. We propose a simple and reliable methodology for the assessment of CXL treatment by using an established rheologic protocol based on small amplitude oscillatory shear (SAOS) measurements. It provides information on the average cross-link density and the elastic modulus of treated cornea samples. Three fresh postmortem porcine corneas were used to study the feasibility of the technique, one serving as control and two receiving corneal collagen cross-linking treatment. Subsequently, five pairs of fresh postmortem porcine corneas received corneal collagen cross-linking treatment with riboflavin and UVA-irradiation (370 nm; irradiance of 3 mW/cm2) for 30 minutes (Dresden protocol); the contralateral porcine corneas were used as control samples. After the treatment, the linear viscoelastic moduli of the corneal samples were measured using SAOS measurements and the average cross-linking densities extracted. For all cases investigated, the dynamic moduli of the cross-linked corneas were higher compared to those of the corresponding control samples. The increase of the elastic modulus of the treated samples was between 122% and 1750%. The difference was statistically significant for all tested samples (P = 0.018, 2-tailed t-test). We report a simple and accurate methodology for quantifying the effects of cross-linking on porcine corneas treated with the Dresden protocol by means of SAOS measurements in the linear regime. The measured dynamic moduli, elastic and viscous modulus, represent the energy storage and energy dissipation, respectively. Hence, they provide a means to assess the changing physical properties of the cross-linked collagen networks after CXL treatment.

Diffusion of protein through the human cornea.

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Charalel, Resmi A; Engberg, Kristin; Noolandi, Jaan; Cochran, Jennifer R; Frank, Curtis; Ta, Christopher N

2012-01-01

To determine the rate of diffusion of myoglobin and bovine serum albumin (BSA) through the human cornea. These small proteins have hydrodynamic diameters of approximately 4.4 and 7.2 nm, and molecular weights of 16.7 and 66 kDa, for myoglobin and BSA, respectively. Diffusion coefficients were measured using a diffusion chamber where the protein of interest and balanced salt solution were in different chambers separated by an ex vivo human cornea. Protein concentrations in the balanced salt solution chamber were measured over time. Diffusion coefficients were calculated using equations derived from Fick's law and conservation of mass in a closed system. Our experiments demonstrate that the diffusion coefficient of myoglobin is 5.5 ± 0.9 × 10(-8) cm(2)/s (n = 8; SD = 1.3 × 10(-8) cm(2)/s; 95% CI: 4.6 × 10(-8) to 6.4 × 10(-8) cm(2)/s) and the diffusion coefficient of BSA is 3.1 ± 1.0 × 10(-8) cm(2)/s (n = 8; SD = 1.4 × 10(-8) cm(2)/s; 95% CI: 2.1 × 10(-8) to 4.1 × 10(-8) cm(2)/s). Our study suggests that molecules as large as 7.2 nm may be able to passively diffuse through the human cornea. With applications in pharmacotherapy and the development of an artificial cornea, further experiments are warranted to fully understand the limits of human corneal diffusion and its clinical relevance. Copyright © 2012 S. Karger AG, Basel.

Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

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Lee, Koon-Ja; Lee, Ji-Young; Lee, Sung Ho; Choi, Tae Hoon

2013-01-01

To understand the corneal regeneration induced by bevacizumab, we investigated the structure changes of stroma and basement membrane regeneration. A Stick soaked in 0.5 N NaOH onto the mouse cornea and 2.5 mg/ml of bevacizumab was delivered into an alkali-burned cornea (2 μl) by subconjunctival injections at 1 hour and 4 days after injury. At 7 days after injury, basement membrane regeneration was observed by transmission electron microscope. Uneven and thin epithelial basement membrane, light density of hemidesmosomes, and edematous collagen fibril bundles are shown in the alkali-burned cornea. Injured epithelial basement membrane and hemidesmosomes and edematous collagen fibril bundles resulting from alkali-burned mouse cornea was repaired by bevacizumab treatment. This study demonstrates that bevacizumab can play an important role in wound healing in the cornea by accelerating the reestablishment of basement membrane integrity that leads to barriers for scar formation. [BMB Reports 2013; 46(4): 195-200] PMID:23615260

Dendritic cells in the cornea during Herpes simplex viral infection and inflammation.

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Kwon, Min S; Carnt, Nicole A; Truong, Naomi R; Pattamatta, Ushasree; White, Andrew J; Samarawickrama, Chameen; Cunningham, Anthony L

2017-11-10

Herpes simplex keratitis is commonly caused by Herpes simplex virus type 1, which primarily infects eyelids, corneas, or conjunctiva. Herpes simplex virus type 1-through sophisticated interactions with dendritic cells (DCs), a type of antigen-presenting cell)-initiates proinflammatory responses in the cornea. Corneas were once thought to be an immune-privileged region; however, with the recent discovery of DCs that reside in the cornea, this long-held conjecture has been overturned. Therefore, evaluating the clinical, preclinical, and cell-based studies that investigate the roles of DCs in corneas infected with Herpes simplex virus is critical. With in vivo confocal microscopy, animal models, and cell culture experiments, we may further the understanding of the sophisticated interactions of Herpes simplex virus with DCs in the cornea and the molecular mechanism associated with it. It has been shown that specific differentiation of DCs using immunohistochemistry, flow cytometry, and polymerase chain reaction analysis in both human and mice tissues and viral tissue infections are integral to increasing understanding. As for in vivo confocal microscopy, it holds promise as it is the least invasive and a real-time investigation. These tools will facilitate the discovery of various targets to develop new treatments. Copyright © 2017 Elsevier Inc. All rights reserved.

Reconstruction of structure and function in tissue engineering of solid organs: Toward simulation of natural development based on decellularization.

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Zheng, Chen-Xi; Sui, Bing-Dong; Hu, Cheng-Hu; Qiu, Xin-Yu; Zhao, Pan; Jin, Yan

2018-04-27

Failure of solid organs, such as the heart, liver, and kidney, remains a major cause of the world's mortality due to critical shortage of donor organs. Tissue engineering, which uses elements including cells, scaffolds, and growth factors to fabricate functional organs in vitro, is a promising strategy to mitigate the scarcity of transplantable organs. Within recent years, different construction strategies that guide the combination of tissue engineering elements have been applied in solid organ tissue engineering and have achieved much progress. Most attractively, construction strategy based on whole-organ decellularization has become a popular and promising approach, because the overall structure of extracellular matrix can be well preserved. However, despite the preservation of whole structure, the current constructs derived from decellularization-based strategy still perform partial functions of solid organs, due to several challenges, including preservation of functional extracellular matrix structure, implementation of functional recellularization, formation of functional vascular network, and realization of long-term functional integration. This review overviews the status quo of solid organ tissue engineering, including both advances and challenges. We have also put forward a few techniques with potential to solve the challenges, mainly focusing on decellularization-based construction strategy. We propose that the primary concept for constructing tissue-engineered solid organs is fabricating functional organs based on intact structure via simulating the natural development and regeneration processes. Copyright © 2018 John Wiley & Sons, Ltd.

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

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Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Use of Fourier-Domain Optical Coherence Tomography to Evaluate Anterior Stromal Opacities in Donor Corneas

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Matthew R. Bald

2013-01-01

Full Text Available Purpose. To evaluate Fourier-domain optical coherence tomography (FD-OCT as an adjunct to traditional slit lamp examination of donor corneas with suspected Anterior Stromal Opacities. Methods. Seven corneas suspected of having anterior stromal opacities by slit lamp examination were evaluated with FD-OCT. Each cornea was evaluated to confirm the presence of opacity and, if present, the depth of opacity was measured. Results. The opacity depth ranged from 82 μm to 624 μm. The initial slit lamp impressions of five of the seven corneas were confirmed by OCT. In two corneas, the OCT findings were different from the initial slit lamp impressions. Slit lamp examination of the first cornea gave the impression of anterior stromal scarring, but OCT showed that the opacity was limited to the epithelium. Slit lamp examination of the second cornea suggested opacity limited to the epithelium, but OCT identified significant sub-Bowman's scarring. In all cases, the Eye Bank Technicians reported that the location and depth of corneal opacity were more sharply defined by OCT than by slit lamp. Conclusion. The high resolution of OCT makes it easier to determine the location of corneal opacities compared to slit lamp examinations. This enhanced visualization can improve decisions regarding transplant suitability of donor corneas.

Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats.

Science.gov (United States)

Sun, Fei; Zhou, Ke; Mi, Wen-juan; Qiu, Jian-hua

2011-11-01

Natural biological conduits containing seed cells have been widely used as an alternative strategy for nerve gap reconstruction to replace traditional nerve autograft techniques. The purpose of this study was to investigate the effects of a decellularized allogeneic artery conduit containing autologous transdifferentiated adipose-derived stem cells (dADSCs) on an 8-mm facial nerve branch lesion in a rat model. After 8 weeks, functional evaluation of vibrissae movements and electrophysiological assessment, retrograde labeling of facial motoneurons and morphological analysis of regenerated nerves were performed to assess nerve regeneration. The transected nerves reconstructed with dADSC-seeded artery conduits achieved satisfying regenerative outcomes associated with morphological and functional improvements which approached those achieved with Schwann cell (SC)-seeded artery conduits, and superior to those achieved with artery conduits alone or ADSC-seeded artery conduits, but inferior to those achieved with nerve autografts. Besides, numerous transplanted PKH26-labeled dADSCs maintained their acquired SC-phenotype and myelin sheath-forming capacity inside decellularized artery conduits and were involved in the process of axonal regeneration and remyelination. Collectively, our combined use of decellularized allogeneic artery conduits with autologous dADSCs certainly showed beneficial effects on nerve regeneration and functional restoration, and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects. Copyright © 2011 Elsevier Ltd. All rights reserved.

Preparation of a nano- and micro-fibrous decellularized scaffold seeded with autologous mesenchymal stem cells for inguinal hernia repair

Directory of Open Access Journals (Sweden)

Zhang Y

2017-02-01

Full Text Available Yinlong Zhang,1,* Yuanyuan Zhou,1,* Xu Zhou,2,* Bin Zhao,1,* Jie Chai,1 Hongyi Liu,1 Yifei Zheng,1 Jinling Wang,3 Yaozong Wang,4 Yilin Zhao2 1Medical College, Xiamen University, 2Department of Oncology and Vascular Intervention Radiology, 3Department of Emergency, 4Department of Orthopaedics, Zhongshan Hospital, Xiamen University, Xiamen, People’s Republic of China *These authors contributed equally to this work Abstract: Prosthetic meshes used for hernioplasty are usually complicated with chronic pain due to avascular fibrotic scar or mesh shrinkage. In this study, we developed a tissue-engineered mesh (TEM by seeding autologous bone marrow-derived mesenchymal stem cells onto nanosized fibers decellularized aorta (DA. DA was achieved by decellularizing the aorta sample sequentially with physical, mechanical, biological enzymatic digestion, and chemical detergent processes. The tertiary structure of DA was constituted with micro-, submicro-, and nanosized fibers, and the original strength of fresh aorta was retained. Inguinal hernia rabbit models were treated with TEMs or acellular meshes (AMs. After implantation, TEM-treated rabbit models showed no hernia recurrence, whereas AM-treated animals displayed bulges in inguinal area. At harvest, TEMs were thicker, have less adhesion, and have stronger mechanical strength compared to AMs (P<0.05. Moreover, TEM showed better cell infiltration, tissue regeneration, and neovascularization (P<0.05. Therefore, these cell-seeded DAs with nanosized fibers have potential for use in inguinal hernioplasty. Keywords: nanobiomaterial, tissue engineering, inguinal hernia, hernioplasty, decellularized aorta 

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold.

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Hassanein, Wessam; Uluer, Mehmet C; Langford, John; Woodall, Jhade D; Cimeno, Arielle; Dhru, Urmil; Werdesheim, Avraham; Harrison, Joshua; Rivera-Pratt, Carlos; Klepfer, Stephen; Khalifeh, Ali; Buckingham, Bryan; Brazio, Philip S; Parsell, Dawn; Klassen, Charlie; Drachenberg, Cinthia; Barth, Rolf N; LaMattina, John C

2017-01-02

Recent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

Thick keratoconic cornea associated with posterior polymorphous corneal dystrophy.

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Zaarour, K; Slim, E; Antoun, J; Waked, N

2017-03-01

We herein report a case of bilateral unusually thick non-edematous keratoconic corneas with associated endothelial features of posterior polymorphous corneal dystrophy (PPCD). We report the case of a 27-year-old myopic woman who presented for refractive surgery. Slit lamp exam showed bilateral corneal protrusion with diffuse deep stromal and endothelial vesicular opacities and small paracentral bands. Topography showed generalized advanced corneal steepening in both eyes with increased anterior and posterior central corneal elevations in comparison to the best fit sphere. Ultrasound pachymetry showed central corneal thickness of 605μm (RE) and 612μm (LE). On specular biomicroscopy, cell density of 2503 cells/mm 2 RE and 1526 cells/mm 2 LE with significant cellular pleomorphism and polymegathism were noted. Clinical and paraclinical findings together suggest the presence of simultaneous keratoconus and PPCD. The literature has suggested an association between PPCD and steep cornea. Moreover, many reports have also described cases of associated PPCD and keratoconus with characteristic thinning and ectasia, in comparison to the unusual thick corneas noted in our patient, despite the absence of edema. Identification of genetics factors is further needed to clarify this association. This case describes a patient whose corneas present features of both keratoconus and PPCD and is unique due to the presence of increased corneal thickness despite the absence of edema. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Correlation between epithelial thickness in normal corneas, untreated ectatic corneas, and ectatic corneas previously treated with CXL; is overall epithelial thickness a very early ectasia prognostic factor?

Science.gov (United States)

Kanellopoulos, Anastasios John; Aslanides, Ioannis M; Asimellis, George

2012-01-01

To determine and correlate epithelial corneal thickness (pachymetric) measurements taken with a digital arc scanning very high frequency ultrasound biomicroscopy (HF UBM) imaging system (Artemis-II), and compare mean and central epithelial thickness among normal eyes, untreated keratoconic eyes, and keratoconic eyes previously treated with collagen crosslinking (CXL). Epithelial pachymetry measurements (topographic mapping) were conducted on 100 subjects via HF UBM. Three groups of patients were included: patients with normal eyes (controls), patients with untreated keratoconic eyes, and patients with keratoconic eyes treated with CXL. Central, mean, and peripheral corneal epithelial thickness was examined for each group, and a statistical study was conducted. Mean, central, and peripheral corneal epithelial thickness was compared between the three groups of patients. Epithelium thickness varied substantially in the keratoconic group, and in some cases there was a difference of up to 20 μm between various points of the same eye, and often a thinner epithelium coincided with a thinner cornea. However, on average, data from the keratoconic group suggested an overall thickening of the epithelium, particularly over the pupil center of the order of +3 μm, while the mean epithelium thickness was on average +1.1 μm, compared to the control population (P = 0.005). This overall thickening was more pronounced in younger patients in the keratoconic group. Keratoconic eyes previously treated with CXL showed, on average, virtually the same average epithelium thickness (mean -0.7 μm, -0.2 μm over the pupil center, -0.9 μm over the peripheral zone) as the control group. This finding further reinforces our novel theory of the "reactive" component of epithelial thickening in corneas that are biomechanically unstable, becoming stable when biomechanical rigidity is accomplished despite persistence of cornea topographic irregularity. A highly irregular epithelium may be

Comparison of the characteristics in hen and quail corneas as experimental models of refractive surgery.

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Gonçalves, G C; Pérez-Merino, P; Martínez-García, M C; Barcía, A; Merayo-Loves, J

2016-07-01

To compare the histological, morphological and the biophysical measurements between hen and quail corneas, in order to determine which of them were better suited for use as an animal model for research into corneal refractive surgery. A study was performed using the biophysical measurements of the cornea (curvature, thickness, refraction, and axial length) of 20 animals (10 hens and 10 quails). The corneas were then prepared for histological analysis under microscopy light. The analysis showed that both groups have the same number of corneal layers as the human cornea and with an evident Bowman's layer. The thickness of the hen cornea and axial length of the eye, 225.3±18.4μm and 12.8±0.25mm, respectively, were larger than that of the quail (Phen central cornea, 3.65±0.08mm, was greater than that for the quail (Ppower of each cornea was similar. The proportion of total corneal thickness of the hen stroma, 82.6%, was more similar to that of the human than was the quail stroma, 72.5%. Within the hen stroma, the density of keratocytes, 8.57±1.49 per 5,000μm(2), was about half that in the quail stroma (Phen cornea, the stromal thickness and proportional similarity of the corneal layers with human cornea, the hen maybe better than the quail as an alternative species suitable for use in studies of corneal refractive surgery. Copyright © 2016 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

Factors influencing the contamination rate of human organ-cultured corneas.

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Röck, Daniel; Wude, Johanna; Bartz-Schmidt, Karl U; Yoeruek, Efdal; Thaler, Sebastian; Röck, Tobias

2017-12-01

To assess the influence of donor, environment and storage factors on the contamination rate of organ-cultured corneas, to consider the microbiological species causing corneal contamination and to investigate the corresponding sensitivities. Data from 1340 consecutive donor corneas were analysed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the contamination rate of organ-cultured corneas for transplantation. The mean annual contamination rate was 1.8 ± 0.4% (range: 1.3-2.1%); 50% contaminations were of fungal origin with exclusively Candida species, and 50% contaminations were of bacterial origin with Staphylococcus species being predominant. The cause of donor death including infection and multiple organ dysfunction syndrome increased the risk of bacterial or fungal contamination during organ culture (p = 0.007 and p = 0.014, respectively). Differentiating between septic and aseptic donors showed an increased risk of contamination for septic donors (p = 0.0020). Mean monthly temperature including warmer months increased the risk of contamination significantly (p = 0.0031). Sex, donor age, death to enucleation, death to corneoscleral disc excision and storage time did not increase the risk of contamination significantly. The genesis of microbial contamination in organ-cultured donor corneas seems to be multifactorial. The main source of fungal or bacterial contamination could be resident species from the skin flora. The rate of microbial contamination in organ-cultured donor corneas seems to be dependent on the cause of donor death and mean monthly temperature. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

In vitro confocal imaging of the rabbit cornea.

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Masters, B R; Paddock, S

1990-05-01

We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin-specific compound fluorescein phalloidin.

Toxicity of methods of implant material sterilization on corneal endothelium

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Singh, G.; Boehnke, Mv.; von Domarus, D.; Draeger, J.

1985-11-01

The toxicity of different procedures utilized for the sterilization of intraocular implant material was assessed on the endothelium of organ-cultured porcine corneas. Polymethylmethacrylate lenses sterilized by treatment with sodium hydroxide (NaOH), ethylene oxide, formaldehyde, and gamma radiation were added to a culture medium containing normal porcine corneas. Considering the viability of endothelial cells, appearance of intracellular degenerative vacuoles, and denudation of corneal Descemet's membrane as criterion for the evaluation of toxicity of different methods of sterilization, the NaOH-treated lenses were found to be the least toxic to porcine corneal endothelium. Phase-contrast microscopy and vital staining of the endothelium permitted direct viewing of the endothelium aiding in the assessment of toxicity.

Lens regeneration from the cornea requires suppression of Wnt/β-catenin signaling.

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Hamilton, Paul W; Sun, Yu; Henry, Jonathan J

2016-04-01

The frog, Xenopus laevis, possesses a high capacity to regenerate various larval tissues, including the lens, which is capable of complete regeneration from the cornea epithelium. However, the molecular signaling mechanisms of cornea-lens regeneration are not fully understood. Previous work has implicated the involvement of the Wnt signaling pathway, but molecular studies have been very limited. Iris-derived lens regeneration in the newt (Wolffian lens regeneration) has shown a necessity for active Wnt signaling in order to regenerate a new lens. Here we provide evidence that the Wnt signaling pathway plays a different role in the context of cornea-lens regeneration in Xenopus. We examined the expression of frizzled receptors and wnt ligands in the frog cornea epithelium. Numerous frizzled receptors (fzd1, fzd2, fzd3, fzd4, fzd6, fzd7, fzd8, and fzd10) and wnt ligands (wnt2b.a, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7b, wnt10a, wnt11, and wnt11b) are expressed in the cornea epithelium, demonstrating that this tissue is transcribing many of the ligands and receptors of the Wnt signaling pathway. When compared to flank epithelium, which is lens regeneration incompetent, only wnt11 and wnt11b are different (present only in the cornea epithelium), identifying them as potential regulators of cornea-lens regeneration. To detect changes in canonical Wnt/β-catenin signaling occurring within the cornea epithelium, axin2 expression was measured over the course of regeneration. axin2 is a well-established reporter of active Wnt/β-catenin signaling, and its expression shows a significant decrease at 24 h post-lentectomy. This decrease recovers to normal endogenous levels by 48 h. To test whether this signaling decrease was necessary for lens regeneration to occur, regenerating eyes were treated with either 6-bromoindirubin-3'-oxime (BIO) or 1-azakenpaullone - both activators of Wnt signaling - resulting in a significant reduction in the percentage of cases with successful

Surveillance of Vittaforma corneae in hot springs by a small-volume procedure.

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Chen, Jung-Sheng; Hsu, Tsui-Kang; Hsu, Bing-Mu; Huang, Tung-Yi; Huang, Yu-Li; Shaio, Men-Fang; Ji, Dar-Der

2017-07-01

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence

[Cornea bank of Lyon: from quality diagnosis to ISO 9001 certification].

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Pascal, P; Chalochet, A; Damour, O

2001-12-01

The tissue and cell bank of the HCL (Hospices Civils de Lyon) has, since 10 June 1999, consisted of two sections with related activities: cell culture for the Skin Substitutes Laboratory (Laboratoire des Substituts Cutanés, LSC) and preservation of corneas at 31 degrees C for the Cornea Bank. As the LSC had been ISO 9001 certified since March 1997 our aim, since merger, was to raise the Cornea Bank to the same level of quality as the LSC, so as to coincide with the renewal of the LSC certificate in February 2000. The methods we used (project, quality control, analysis and process optimization) led us to receive official certification only nine months after the merger. The procedure started with a program of quality control at the Cornea Bank from February 1999 onwards, in order to list the work and equipment required, evaluate its documentation system and what was needed to incorporate this new activity into the existing system of quality assurance at the LSC. On the 7th March 2000, the Tissue and Cell Bank of the HCL obtained an ISO 9001 certificate for its combined functions. As well as achieving our objectives and the strong points highlighted by the auditor during the renewal process, this quality assessment revealed many advantages: improvements in the conservation of corneas, economies in staff replacement and reductions in both the cost of maintaining quality, the cost of the corneas themselves, etc. The decree 'Banque' no. 99-741 of 30th August 1999, which put in place the system of authorization of tissue banks in France, demands quality control. Our application for certification which started in early 1999 had anticipated this regulation. This helped us enormously when compiling the dossier accompanying the official request and was an essential element in obtaining the favourable response of the ASSAPS on 21 June 2000.

Accelerating repaired basement membrane after bevacizumab treatment on alkali-burned mouse cornea

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Koon-Ja Lee

2013-04-01

Full Text Available To understand the corneal regeneration induced by bevacizumab,we investigated the structure changes of stroma andbasement membrane regeneration. A Stick soaked in 0.5 NNaOH onto the mouse cornea and 2.5 mg/ml of bevacizumabwas delivered into an alkali-burned cornea (2 μl by subconjunctivalinjections at 1 hour and 4 days after injury. At 7 daysafter injury, basement membrane regeneration was observedby transmission electron microscope. Uneven and thin epithelialbasement membrane, light density of hemidesmosomes,and edematous collagen fibril bundles are shown in thealkali-burned cornea. Injured epithelial basement membraneand hemidesmosomes and edematous collagen fibril bundlesresulting from alkali-burned mouse cornea was repaired bybevacizumab treatment. This study demonstrates that bevacizumabcan play an important role in wound healing in thecornea by accelerating the reestablishment of basementmembrane integrity that leads to barriers for scar formation.[BMB Reports 2013; 46(4: 195-200

Cornea nerve fiber quantification and construction of phenotypes in patients with fibromyalgia.

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Oudejans, Linda; He, Xuan; Niesters, Marieke; Dahan, Albert; Brines, Michael; van Velzen, Monique

2016-03-23

Cornea confocal microscopy (CCM) is a novel non-invasive method to detect small nerve fiber pathology. CCM generally correlates with outcomes of skin biopsies in patients with small fiber pathology. The aim of this study was to quantify the morphology of small nerve fibers of the cornea of patients with fibromyalgia in terms of density, length and branching and further phenotype these patients using standardized quantitative sensory testing (QST). Small fiber pathology was detected in the cornea of 51% of patients: nerve fiber length was significantly decreased in 44% of patients compared to age- and sex-matched reference values; nerve fiber density and branching were significantly decreased in 10% and 28% of patients. The combination of the CCM parameters and sensory tests for central sensitization, (cold pain threshold, mechanical pain threshold, mechanical pain sensitivity, allodynia and/or windup), yielded four phenotypes of fibromyalgia patients in a subgroup analysis: one group with normal cornea morphology without and with signs of central sensitization, and a group with abnormal cornea morphology parameters without and with signs of central sensitization. In conclusion, half of the tested fibromyalgia population demonstrates signs of small fiber pathology as measured by CCM. The four distinct phenotypes suggest possible differences in disease mechanisms and may require different treatment approaches.

Sorption of sodium hydroxide by type I collagen and bovine corneas.

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Whikehart, D R; Edwards, W C; Pfister, R R

1991-01-01

There are no quantitative studies on the uptake of alkali into corneal tissues. To study this phenomenon, both type I collagen and bovine corneas were incubated in sodium hydroxide (NaOH) under varying conditions for periods up to 27.5 h. The sorption (absorption or adsorption) of the alkali to protein and tissue was measured as the quantity of NaOH no longer available for titration to neutrality with hydrochloric acid. Sorption was found to be dependent on the concentration of NaOH (0.01-1 N) but independent of the incubation temperature (4-35 degrees C). In whole cornea, sorption of 1 N NaOH began immediately and increased with time up to 6 h. After 6 h, sorption decreased, together with the observed degradation and solubilization of the tissue. Stripping of the corneal endothelium alone or of the endothelium and epithelium increased sorption in a similar manner when compared to whole corneas for periods up to 4 h. These observations are compatible with ionic and nonionic bonding of hydroxide ions to collagen (including that of the cornea) and the subsequent release of hydroxide ions during hydrolysis of the protein itself. Indirect evidence also suggests the inclusion of quantities of unbound hydroxide ions in hydrated gels of glycosaminoglycans. It is proposed that in a chemical burn of the cornea, alkali is both stored in the tissue (by sorption) and reacted with it (by hydrolysis), without any net consumption of alkali taking place.

Intrastromal Injection of China Painting Ink in Corneas of Male Rabbits: Clinical and Histological Study.

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Alsmman Hassan, Alahmady Hamad; Abd Elhaliem Soliman, Nesreen Gamal-Eldeen

2016-01-01

Background. Many patients with corneal opacity or complicated cataract in blind eye ask for cosmoses. In this study we tried to investigate the staining of corneas of male rabbits by Rotring China painting ink and to study the histological changes. Method. 10 eyes of 10 male Baladi Egyptian rabbits were injected (0.1 mL) intrastromally in the cornea by the use of China painting ink (Rotring Tinta China) through insulin syringe (27-gauge needle) by single injection; clinical follow-up is for 6 months and lastly the rabbits were scarified and the stained eyes were enucleated for histological analysis. Results. Clinically the stain was stable in color and distribution in corneas with no major complications. Histological results of the stained rabbit corneas showed blackish pigmentation in the corneal stroma without any inflammatory cellular infiltration. Some fibroblast cells had pigment granules in their cytoplasm in the adjacent layers. Conclusion. Corneal staining by China painting ink is effective and safe in staining of male rabbits cornea; however further study in human corneas with longer follow-up period is advisable.

Intrastromal Injection of China Painting Ink in Corneas of Male Rabbits: Clinical and Histological Study

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Alahmady Hamad Alsmman Hassan

2016-01-01

Full Text Available Background. Many patients with corneal opacity or complicated cataract in blind eye ask for cosmoses. In this study we tried to investigate the staining of corneas of male rabbits by Rotring China painting ink and to study the histological changes. Method. 10 eyes of 10 male Baladi Egyptian rabbits were injected (0.1 mL intrastromally in the cornea by the use of China painting ink (Rotring Tinta China through insulin syringe (27-gauge needle by single injection; clinical follow-up is for 6 months and lastly the rabbits were scarified and the stained eyes were enucleated for histological analysis. Results. Clinically the stain was stable in color and distribution in corneas with no major complications. Histological results of the stained rabbit corneas showed blackish pigmentation in the corneal stroma without any inflammatory cellular infiltration. Some fibroblast cells had pigment granules in their cytoplasm in the adjacent layers. Conclusion. Corneal staining by China painting ink is effective and safe in staining of male rabbits cornea; however further study in human corneas with longer follow-up period is advisable.

Hydration and transparency of the rabbit cornea irradiated with UVB-doses of 0.25 J/cm(2) and 0.5 J/cm(2) compared with equivalent UVB radiation exposure reaching the human cornea from sunlight.

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Cejka, Cestmír; Ardan, Taras; Sirc, Jakub; Michálek, Jiří; Beneš, Jiří; Brůnová, Blanka; Rosina, Jozef

2011-07-01

Exposure of the cornea to UV radiation from sunlight evokes intraocular inflammation, photokeratitis. Photokeratitis is caused by UVB radiation. It is accompanied by changes of corneal hydration and light absorption. The aim of this study was to examine the effect of two UVB doses on corneal optics in rabbits and to compare these UVB doses with the equivalent exposure of UVB radiation reaching the human cornea from sunlight. Rabbit corneas were irradiated with a daily UVB dose of 0.25 J/cm(2) or 0.5 J/cm(2) for 4 days. One day after finishing the irradiations the rabbits were sacrificed and corneal light absorption measured using our spectrophotometrical method. Corneal hydration was examined using an ultrasonic Pachymeter every experimental day before the irradiation procedure and the last day before sacrificing the animals. Changes in corneal optics appeared after the repeated exposure of the cornea to a UVB dose of 0.25 J/ cm(2) and massively increased after the repeated exposure of the cornea to a UVB dose of 0.5 J/cm(2). The first significant changes in corneal hydration appeared after a single exposure of the cornea to a UVB dose of 0.25 J/cm(2). Changes in corneal hydration appeared after the exposure of the rabbit cornea to a single UVB dose equivalent to 2.6 hours of solar UVB radiation reaching the human cornea, as measured by UVB sensors embedded in the eyes of mannequin heads facing the sun on a beach at noon in July. Repeated exposure of the rabbit cornea to the same UVB dose evoked profound changes in corneal optics. Although comparison of experimental and outdoor conditions are only approximate, the results in rabbits point to the danger for the human eye from UVB radiation when short stays in sunlight are repeated for several consecutive days without UV protection.

Lectin receptors in the human cornea.

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Holmes, M J; Mannis, M J; Lund, J; Jacobs, L

Five different biotin labeled lectins, Concanavalin-A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA1), Ulex europaeus agglutinin (UEA1), and soybean agglutinin (SBA) were used to study lectin receptors on formalin-fixed paraffin embedded human corneas. Con A stained the cytoplasm, cell, and nuclear membranes of the epithelial cells and stained the stroma diffusely. WGA stained the superficial epithelial cells, the epithelial cell membranes, and the keratocytes of the stroma. SBA did not react with any of the corneal layers. RCA1 heavily stained the keratocytes but did not stain the epithelium. UEA1 lightly stained the epithelial cell cytoplasm and interstitial stroma. All staining reactions could be abolished by omission of the lectin or by the use of the appropriate inhibitory sugar. The lectin binding patterns reported here provide a means for further investigation of carbohydrate structures in the human cornea in both normal and disease states.

Elastic microfibril distribution in the cornea: Differences between normal and keratoconic stroma.

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White, Tomas L; Lewis, Philip N; Young, Robert D; Kitazawa, Koji; Inatomi, Tsutomu; Kinoshita, Shigeru; Meek, Keith M

2017-06-01

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae provides the tissue with tensile strength, whilst the spatial arrangement of individual collagen fibrils within the lamellae confers transparency. In keratoconus, this precise stromal arrangement is lost, resulting in ectasia and visual impairment. In the normal cornea, we previously characterised the three-dimensional arrangement of an elastic fiber network spanning the posterior stroma from limbus-to-limbus. In the peripheral cornea/limbus there are elastin-containing sheets or broad fibers, most of which become microfibril bundles (MBs) with little or no elastin component when reaching the central cornea. The purpose of the current study was to compare this network with the elastic fiber distribution in post-surgical keratoconic corneal buttons, using serial block face scanning electron microscopy and transmission electron microscopy. We have demonstrated that the MB distribution is very different in keratoconus. MBs are absent from a region of stroma anterior to Descemet's membrane, an area that is densely populated in normal cornea, whilst being concentrated below the epithelium, an area in which they are absent in normal cornea. We contend that these latter microfibrils are produced as a biomechanical response to provide additional strength to the anterior stroma in order to prevent tissue rupture at the apex of the cone. A lack of MBs anterior to Descemet's membrane in keratoconus would alter the biomechanical properties of the tissue, potentially contributing to the pathogenesis of the disease. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cytogenetic damage and postradiation restoration of eye cornea epithelium of Rodentia characterizing by different radiosensitivity

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Popova, M.F.; Bulyakova, N.V.

1983-01-01

Intensity of beam damage and reparation of eye cornea epithelium of animals inhabiting under different conditions and differing by radiosensitivity has been studied. Mice differing by high radiosensitivity have the hardest cytogenetic damage. Cornea epithelium of bank voles is more radiostable than that of mice. The most negligible damages of cornea epithelium is observed in Mongolian sandwort despite the fact that their total radiation stability is lower than that of bank voles. High protective-restoring properties of eye cornea epithelium of Mongolian sandwort are explained by the structure of epithelium cells diffe-- ring by a large number of cytoplasm

Porcine endogenous retroviral nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant.

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Binette, Tanya M; Seeberger, Karen L; Lyon, James G; Rajotte, Ray V; Korbutt, Gregory S

2004-07-01

Porcine islets represent an alternative source of insulin-producing tissue, however, porcine endogenous retrovirus (PERV) remains a concern. In this study, SCID mice were transplanted with nonencapsulated (non-EC), microencapsulated (EC) or macroencapsulated (in a TheraCyte trade mark device) neonatal porcine islets (NPIs), and peripheral tissues were screened for presence of viral DNA and mRNA. To understand the role of an intact immune system in PERV incidence, mice with established NPI grafts were reconstituted with splenocytes. Peripheral tissues were screened for PERV and porcine DNA using PCR. Tissues with positive DNA were analyzed for PERV mRNA using RT-PCR. No significant difference was observed between non-EC and EC transplants regarding presence of PERV or porcine-specific DNA or mRNA. In reconstituted animals, little PERV or porcine DNA, and no PERV mRNA was detected. No PERV or porcine-specific DNA was observed in animals implanted with a TheraCyte trade mark device. In conclusion, an intact immune system significantly lowered the presence of PERV. Microencapsulation of islets did not alter PERV presence, however, macroencapsulation in the TheraCyte device did. Lower PERV incidence coincided with lower porcine DNA in peripheral tissues, linking the presence of PERV to migration of porcine cells.

Preparation and Characterization of a Novel Decellularized Fibrocartilage "Book" Scaffold for Use in Tissue Engineering.

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Liyun Guo

Full Text Available At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm with 10 layers, each layer (akin to a page of a book with a thickness of 100-μm. These fibrocartilage "book" scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel "book" fibrocartilage scaffold could have potential applications in tissue engineering.

Preparation and Characterization of a Novel Decellularized Fibrocartilage "Book" Scaffold for Use in Tissue Engineering.

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Guo, Liyun; Qu, Jin; Zheng, Cheng; Cao, Yong; Zhang, Tao; Lu, Hongbin; Hu, Jianzhong

2015-01-01

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage "book" scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel "book" fibrocartilage scaffold could have potential applications in tissue engineering.

In vivo confocal Raman spectroscopy of the human cornea.

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Bauer, N J; Hendrikse, F; March, W F

1999-07-01

To investigate the feasibility of a confocal Raman spectroscopic technique for the noninvasive assessment of corneal hydration in vivo in two legally blind subjects. A laser beam (632.8 nm; 15 mJ) was maintained on the cornea by using a microscope objective lens (x25 magnification, NA = 0.5, f = 10 mm) both for focusing the incident light as well as collecting the Raman backscattered light, in a 180 degrees backscatter configuration. An optical fiber, acting as the confocal pinhole for elimination of light from out-of-focus places, was coupled to a spectrometer that dispersed the collected light onto a sensitive array detector for rapid spectral data acquisition over a range from 2,890 to 3,590/cm(-1). Raman spectra were recorded from the anterior 100-150 microm of the cornea over a period before and after topical application of a mild dehydrating solution. The ratio between the amplitudes of the signals at 3,400/cm(-1) (OH-vibrational mode of water) and 2,940/cm(-1) (CH-vibrational mode of proteins) was used as a measure for corneal hydration. High signal-to-noise ratio (SNR = 25) Raman spectra were obtained from the human corneas by using 15 mJ of laser light energy. Qualitative changes in the hydration of the anteriormost part of the corneas could be observed as a result of the dehydrating agent. With adequate improvements in system safety, confocal Raman spectroscopy could potentially be applied clinically as a noninvasive tool for the assessment of corneal hydration in vivo.

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

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Masihzadeh, Omid; Ammar, David A.; Kahook, Malik Y.; Gibson, Emily A.; Lei, Tim C.

2013-03-01

The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

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Jorge S Burns

Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

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Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

Experimental corneal calcification, hydration and /sup 45/Ca uptake in rabbit corneas incubated in vitro

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Obenberger, J [Ceskoslovenska Akademie Ved, Prague. Oftalmologicka Laborator; Dobiasova, M; Babicky, A [Ceskoslovenska Akademie Ved, Prague. Isotopova Laborator Biologickych Ustavu

1974-07-01

Experimental corneal calcification could easily be produced by a combination of corneal injury (perfusion of the anterior chamber of the eye with a solution of potassium permanganate) amd dihydrotachysterol (DHT) treatment. Rabbit corneas with induced calcification as well as corneas of three additional groups of rabbits, i.e. those treated with permanganate or DHT only and control animals were incubated for two hours in a medium containing /sup 45/Ca. An increased uptake of /sup 45/Ca into the cornea was found in the group of rabbits receiving DHT only. Potassium cyanide added to the incubation medium did not affect corneal hydration nor the final activity of the incubated corneas. (auth)

Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

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Funari, Vincent A; Winkler, Michael; Brown, Jordan; Dimitrijevich, Slobodan D; Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

2013-01-01

MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

Differentially expressed wound healing-related microRNAs in the human diabetic cornea.

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Vincent A Funari

Full Text Available MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6 and diabetic (n=6 central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR and by in situ hybridization (ISH in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR or their inhibitors (antagomirs using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

Ex vivo rabbit cornea diffusion studies with a soluble insert of moxifloxacin.

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Sebastián-Morelló, María; Calatayud-Pascual, María Aracely; Rodilla, Vicent; Balaguer-Fernández, Cristina; López-Castellano, Alicia

2018-02-01

The objective of this research was to develop and evaluate an ocular insert for the controlled drug delivery of moxifloxacin which could perhaps be used in the treatment of corneal keratitis or even bacterial endophthalmitis. We have evaluated the ex vivo ocular diffusion of moxifloxacin through rabbit cornea, both fresh and preserved under different conditions. Histological studies were also carried out. Subsequently, drug matrix inserts were prepared using bioadhesive polymers. The inserts were evaluated for their physicochemical parameters. Ophthalmic ex vivo permeation of moxifloxacin was carried out with the most promising insert. The formulate insert was thin and provided higher ocular diffusion than commercial formulations. Ocular diffusion studies revealed significant differences between fresh and frozen corneas. Histological examinations also showed differences in the thickness of stroma between fresh and frozen corneas. The ophthalmic insert we have developed allows a larger quantity of moxifloxacin to permeate through the cornea than existing commercial formulations of the drug. Ocular delivery of moxifloxacin with this insert could be a new approach for the treatment of eye diseases.

Inflammation Modulatory Protein TSG-6 for Chemical Injuries to the Cornea

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-14-1-0495 TITLE: Inflammation Modulatory Protein TSG-6 for Chemical Injuries to the Cornea PRINCIPAL INVESTIGATOR...2017 4. TITLE AND SUBTITLE Inflammation Modulatory Protein TSG-6 for Chemical Injuries to the Cornea Infla I Inflammation Modulatory Protein TSG-6...anti-inflammatory protein , TSG-6, which has a novel mechanism of action. Chemical injuries of the eye are difficult to treat, and may lead to

High-resolution, label-free two-photon imaging of diseased human corneas

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Batista, Ana; Breunig, Hans Georg; König, Aisada; Schindele, Andreas; Hager, Tobias; Seitz, Berthold; König, Karsten

2018-03-01

The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells' metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells' morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells' metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.

Oxygen transport through soft contact lens and cornea: Lens characterization and metabolic modeling

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Chhabra, Mahendra

The human cornea requires oxygen to sustain metabolic processes critical for its normal functioning. Any restriction to corneal oxygen supply from the external environment (e.g., by wearing a low oxygen-permeability contact lens) can lead to hypoxia, which may cause corneal edema (swelling), limbal hyperemia, neovascularization, and corneal acidosis. The need for adequate oxygen to the cornea is a major driving force for research and development of hypertransmissible soft contact lenses (SCLs). Currently, there is no standard technique for measuring oxygen permeability (Dk) of hypertransmissible silicone-hydrogel SCLs. In this work, an electrochemistry-based polarographic apparatus was designed, built, and operated to measure oxygen permeability in hypertransmissible SCLs. Unlike conventional methods where a range of lens thickness is needed for determining oxygen permeabilities of SCLs, this apparatus requires only a single lens thickness. The single-lens permeameter provides a reliable, efficient, and economic tool for measuring oxygen permeabilities of commercial hypertransmissible SCLs. The single-lens permeameter measures not only the product Dk, but, following modification, it measures separately diffusivity, D, and solubility, k, of oxygen in hypertransmissible SCLs. These properties are critical for designing better lens materials that ensure sufficient oxygen supply to the cornea. Metabolism of oxygen in the cornea is influenced by contact-lens-induced hypoxia, diseases such as diabetes, surgery, and drug treatment, Thus, estimation of the in-vivo corneal oxygen consumption rate is essential for gauging adequate oxygen supply to the cornea. Therefore, we have developed an unsteady-state reactive-diffusion model for the cornea-contact-lens system to determine in-vivo human corneal oxygen-consumption rate. Finally, a metabolic model was developed to determine the relation between contact-lens oxygen transmissibility (Dk/L) and corneal oxygen deficiency. A

Morphology of endothelial cells from different regions of the equine cornea

OpenAIRE

Faganello, Cláudia Skilhan; Silva, Vanessa Ruiz Moura da; Andrade, Maria Cristina Caldart de; Carissimi, André Silva; Pigatto, João Antonio Tadeu

2016-01-01

ABSTRACT: The objective of this study was to evaluate the morphology of different regions of the equine cornea using optical microscopy. Both healthy eyes of eight horses, male or female, of different ages were evaluated. Corneas were stained with alizarin red vital dye and subsequently examined and photographed using optical microscopy. Corneal endothelial morphology of central, superior, inferior, temporal and nasal areas was assessed. One hundred endothelial cells from each corneal area we...

Application of detergents or high hydrostatic pressure as decellularization processes in uterine tissues and their subsequent effects on in vivo uterine regeneration in murine models.

Directory of Open Access Journals (Sweden)

Erna G Santoso

Full Text Available Infertility caused by ovarian or tubal problems can be treated using In Vitro Fertilization and Embryo Transfer (IVF-ET; however, this is not possible for women with uterine loss and malformations that require uterine reconstruction for the treatment of their infertility. In this study, we are the first to report the usefulness of decellularized matrices as a scaffold for uterine reconstruction. Uterine tissues were extracted from Sprague Dawley (SD rats and decellularized using either sodium dodecyl sulfate (SDS or high hydrostatic pressure (HHP at optimized conditions. Histological staining and quantitative analysis showed that both SDS and HHP methods effectively removed cells from the tissues with, specifically, a significant reduction of DNA contents for HHP constructs. HHP constructs highly retained the collagen content, the main component of extracellular matrices in uterine tissue, compared to SDS constructs and had similar content levels of collagen to the native tissue. The mechanical strength of the HHP constructs was similar to that of the native tissue, while that of the SDS constructs was significantly elevated. Transmission electron microscopy (TEM revealed no apparent denaturation of collagen fibers in the HHP constructs compared to the SDS constructs. Transplantation of the decellularized tissues into rat uteri revealed the successful regeneration of the uterine tissues with a 3-layer structure 30 days after the transplantation. Moreover, a lot of epithelial gland tissue and Ki67 positive cells were detected. Immunohistochemical analyses showed that the regenerated tissues have a normal response to ovarian hormone for pregnancy. The subsequent pregnancy test after 30 days transplantation revealed successful pregnancy for both the SDS and HHP groups. These findings indicate that the decellularized matrix from the uterine tissue can be a potential scaffold for uterine regeneration.

The use of human cornea organotypic cultures to study herpes simplex virus type 1 (HSV-1)-induced inflammation.

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Drevets, Peter; Chucair-Elliott, Ana; Shrestha, Priyadarsini; Jinkins, Jeremy; Karamichos, Dimitrios; Carr, Daniel J J

2015-10-01

To determine the utility of human organotypic cornea cultures as a model to study herpes simplex virus type 1 (HSV-1)-induced inflammation and neovascularization. Human organotypic cornea cultures were established from corneas with an intact limbus that were retrieved from donated whole globes. One cornea culture was infected with HSV-1 (10(4) plaque-forming units), while the other cornea from the same donor was mock-infected. Supernatants were collected at intervals post-culture with and without infection to determine viral titer (by plaque assay) and pro-angiogenic and proinflammatory cytokine concentration by suspension array analysis. In some experiments, the cultured corneas were collected and evaluated for HSV-1 antigens by immunohistochemical means. Another set of experiments measured susceptibility of human three-dimensional cornea fibroblast constructs, in the presence and absence of TGF-β1, to HSV-1 infection in terms of viral replication and the inflammatory response to infection as a comparison to the organotypic cornea cultures. Organotypic cornea cultures and three-dimensional fibroblast constructs exhibited varying degrees of susceptibility to HSV-1. Fibroblast constructs were more susceptible to infection in terms of infectious virus recovered in a shorter period of time. There were changes in the levels of select pro-angiogenic or proinflammatory cytokines that were dictated as much by the cultures producing them as by whether they were infected with HSV-1 or treated with TGF-β1. Organotypic cornea and three-dimensional fibroblast cultures are likely useful for the identification and short-term study of novel antiviral compounds and virus replication, but are limited in the study of the local immune response to infection.

7 CFR 1230.611 - Porcine animal.

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2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold to...

Cryo-chemical decellularization of the whole liver for mesenchymal stem cells-based functional hepatic tissue engineering.

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Jiang, Wei-Cheng; Cheng, Yu-Hao; Yen, Meng-Hua; Chang, Yin; Yang, Vincent W; Lee, Oscar K

2014-04-01

Liver transplantation is the ultimate treatment for severe hepatic failure to date. However, the limited supply of donor organs has severely hampered this treatment. So far, great potentials of using mesenchymal stem cells (MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for generation of well-transplantable hepatic tissues. In this study, we utilized a cryo-chemical decellularization method which combines physical and chemical approach to generate acellular liver scaffolds (ALS) from the whole liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage, albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than two-dimensional culture in vitro. Importantly, the hepatic-like tissues (HLT) generated by repopulating ALS with MSCs are able to act as functional grafts and rescue lethal hepatic failure after transplantation in vivo. In summary, the cryo-chemical method used in this study is suitable for decellularization of liver and create acellular scaffolds that can support hepatic differentiation of MSCs and be used to fabricate functional tissue-engineered liver constructs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preparation and Characterization of a Novel Decellularized Fibrocartilage “Book” Scaffold for Use in Tissue Engineering

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Guo, Liyun; Qu, Jin; Zheng, Cheng; Cao, Yong; Zhang, Tao; Lu, Hongbin; Hu, Jianzhong

2015-01-01

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage “book” scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel “book” fibrocartilage scaffold could have potential applications in tissue engineering. PMID:26636672

Cornea procurement, preservation and transplantation

International Nuclear Information System (INIS)

Hudson Silva

1999-01-01

Our experience on this subject is 40 years and we have seen the developments everywhere during this total period. Cornea procurement has been an unsurmountable problem in a large number of countries, mostly due to social and cultural reasons. The requirements for a simple eye bank to manage the supply for a local hospital are minimal. What is the most important is the donor base. In Sri Lanka nurtured in the traditions of Theravada Buddhism the donor base was easy to prepare. We had no difficulties with Christians and Hindus. For Muslims there are Fatwas in favour of donating eyes. But we always found them to be a reluctant group. But in predominantly Muslim countries, it is imperative that social workers should take up this issue, so those thousands may be helped. In all areas where attempts are made to procure comeas, it is important to have a day-and-night service available at short notice to collect any eyes offered. A trained technician with sterilised instruments to obtain eyes and blood sample must be available at a convenient location preferably in a large hospital. Even in places where the supply is infrequent, it is essential to have the readily-sterilised instruments to proceed to a donor site at short notice. To get eyes, general publicity in mass media alone is not sufficient. Such publicity makes people aware of the need for donor eyes and where a technician/doctor is available. What is more important is to canvass personally after a person has died For this purpose trained social workers may be employed, or the technician can do the motivation, as we do in Sri Lanka. Once eyes are removed, it is essential to pay careful attention to the appearance of the cadaver. It should not (and need not) cause any deformity as such event has a profound negative effect on the campaign. Many different preservation methods have been used over the years. Mc Carey-Kaufmann (MK) medium has been the standard for many years, and gives a life span of 4-5 days. The formula

Efficacious and safe tissue-selective controlled gene therapy approaches for the cornea.

Directory of Open Access Journals (Sweden)

Rajiv R Mohan

2011-04-01

Full Text Available Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5, and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×10(12 vg/ml expressing green fluorescent protein gene (GFP was topically applied onto normal or diseased (fibrotic or neovascularized rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point. Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5

Why Does the Healthy Cornea Resist Pseudomonas aeruginosa Infection?

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Evans, David J.; Fleiszig, Suzanne M. J.

2013-01-01

Purpose To provide our perspective on why the cornea is resistant to infection based on our research results with Pseudomonas aeruginosa. Perspective We focus on our current understanding of the interplay between bacteria, tear fluid and the corneal epithelium that determine health as the usual outcome, and propose a theoretical model for how contact lens wear might change those interactions to enable susceptibility to P. aeruginosa infection. Methods Use of “null-infection” in vivo models, cultured human corneal epithelial cells, contact lens-wearing animal models, and bacterial genetics help to elucidate mechanisms by which P. aeruginosa survive at the ocular surface, adheres, and traverses multilayered corneal epithelia. These models also help elucidate the molecular mechanisms of corneal epithelial innate defense. Results and Discussion Tear fluid and the corneal epithelium combine to make a formidable defense against P. aeruginosa infection of the cornea. Part of that defense involves the expression of antimicrobials such as β-defensins, the cathelicidin LL-37, cytokeratin-derived antimicrobial peptides, and RNase7. Immunomodulators such as SP-D and ST2 also contribute. Innate defenses of the cornea depend in part on MyD88, a key adaptor protein of TLR and IL-1R signaling, but the basal lamina represents the final barrier to bacterial penetration. Overcoming these defenses involves P. aeruginosa adaptation, expression of the type three secretion system, proteases, and P. aeruginosa biofilm formation on contact lenses. Conclusion After more than two decades of research focused on understanding how contact lens wear predisposes to P. aeruginosa infection, our working hypothesis places blame for microbial keratitis on bacterial adaptation to ocular surface defenses, combined with changes to the biochemistry of the corneal surface caused by trapping bacteria and tear fluid against the cornea under the lens. PMID:23601656

Anterior segment optical coherence tomography for evaluation of cornea and ocular surface

Directory of Open Access Journals (Sweden)

Mittanamalli S Sridhar

2018-01-01

Full Text Available Current corneal assessment technologies make the process of corneal evaluation extremely fast and simple. Several devices and technologies allow to explore and manage patients better. Optical coherence tomography (OCT technology has evolved over the years, and hence a detailed evaluation of anterior segment (AS structures such as cornea, conjunctiva, tear meniscus, anterior chamber, iris, and crystalline lens has been possible in a noncontact and safe procedure. The purpose of this special issue is to present and update in the evaluation of cornea and ocular surface, and this paper reviews a description of the AS-OCT, presenting the technology and common clinical uses of OCT in the management of diseases involving cornea and ocular surface to provide an updated information of the clinical recommendations of this technique in eye care practice.

[The value of methods for morphofunctional analysis of cornea in cataract surgery].

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Borodina, N B; Kobzova, M V; Musaeva, G M

2011-01-01

The characteristics of morphofunctional status of cornea after extracapsular cataract extraction and phakoemulsification with IOL implantation (30 and 58 operations respectively) were analyzed in detail using up-to-date diagnostic techniques. The results of examination using developed algorithm including study of light transmission, refraction and protective function of cornea show advantage of microinvasive ultrasound technique of cataract surgery in terms of minimal impact on corneal structure, optical and biomechanical characteristics.

Second Harmonic Generation Imaging Analysis of Collagen Arrangement in Human Cornea.

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Park, Choul Yong; Lee, Jimmy K; Chuck, Roy S

2015-08-01

To describe the horizontal arrangement of human corneal collagen bundles by using second harmonic generation (SHG) imaging. Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane. Collagen bundles in the corneal lamellae demonstrated a complex layout merging and splitting within a single lamellar plane. The patterns were significantly different in the superficial and limbal cornea when compared with deep and central regions. Collagen bundles were smaller in the superficial layer and larger in deep lamellae. By using SHG imaging, the horizontal arrangement of corneal collagen bundles was elucidated at different depths and focal regions of the human cornea.

Optical coherent tomography measurements of the diffusion rate of water and drugs in an isolated and whole cornea

International Nuclear Information System (INIS)

Larin, Kirill V; Ghosn, M G

2006-01-01

The passive diffusion of drugs through the epithelial surfaces of an eye (the most widespread method for medical treatment of various diseases) is considered. The permeability of water and drugs through rabbit cornea was measured in the isolated cornea (separate from an eye) and in the whole cornea. The permeability coefficients of water and dexamethasone were estimated by the method of optical coherence tomography (OCT). Because multiple photon scattering introduces noise and distortions to the OCT signal, measurements were performed at depths up to 500 μm where most likely single scattering of light occurs in cornea. It is shown that the permeability coefficients in the isolated and whole cornea strongly differ from each other. For example, the water permeability in the isolated and whole cornea is (7.09±0.12)x10 -5 and (1.71±0.51)x10 -5 cm s -1 , respectively. (special issue devoted to multiple radiation scattering in random media)

Results of aortic valve repair using decellularized bovine pericardium in congenital surgery.

Science.gov (United States)

Nordmeyer, Sarah; Murin, Peter; Schulz, Antonia; Danne, Friederike; Nordmeyer, Johannes; Kretzschmar, Johanna; Sumbadze, Daria; Schmitt, Katharina Rose Luise; Miera, Oliver; Cho, Mi-Young; Sinzobahamvya, Nicodeme; Berger, Felix; Ovroutski, Stanislav; Photiadis, Joachim

2018-04-30

The search for an optimal patch material for aortic valve reconstruction (AVR) is an ongoing challenge. In this study, we report our experience of AVR using decellularized bovine pericardial patch material in congenital heart surgery. Data of 40 consecutive patients who underwent AVR using the CardioCel® patch (Admedus Regen Pty Ltd, Perth, WA, Australia) between February 2014 and August 2016 were retrospectively reviewed. The median age of the patients at operation was 9 (2-34) years, and 18 patients were younger than 7 years. Twenty-six patients initially presented with aortic valve insufficiency (AI) and 14 with stenosis. Clinical and echocardiographic data were available until August 2017 for a median postoperative follow-up (FU) of 22 (6-42) months. Nine of 40 (23%) patients experienced an event during FU (death: n = 1, 2.5%; reoperation: n = 8, 20%). Overall, the probability of freedom from reoperation or death was 97 ± 3%, 76 ± 9% and 57 ± 12% at 12, 24 and 36 months of FU, respectively. Reason for reoperation was stenosis in 3 (37.5%) patients, insufficiency in 4 (50%) patients and 1 (12.5%) patient was diagnosed with aortic valve endocarditis. Of the remaining 31 patients, 2 patients are scheduled for reoperation (aortic valve stenosis: n = 1 and AI: n = 1) and 9 patients exhibit worsening of aortic valve function with moderate AI. Freedom from developing combined end point [death/reoperation/moderate degree of aortic valve dysfunction (aortic valve stenosis, AI)] after AVR was 92 ± 5%, 55 ± 9% and 28 ± 9% at 12, 24 and 36 months, respectively. AVR using decellularized bovine pericardial patch material in patients with congenital aortic valve disease show unsatisfactory results within the first 3 years of FU.

Monoclonal antibodies specific to heat-treated porcine blood.

Science.gov (United States)

Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

2016-05-01

Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Enhancement of skin wound healing with decellularized scaffolds loaded with hyaluronic acid and epidermal growth factor

Energy Technology Data Exchange (ETDEWEB)

Su, Zhongchun; Ma, Huan; Wu, Zhengzheng [Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632 (China); Zeng, Huilan [Department of Hematology, The First Affiliated Hospital, Jinan University, Guangzhou 510632 (China); Li, Zhizhong [Department of Bone, The First Affiliated Hospital, Jinan University, Guangzhou 510632 (China); Wang, Yuechun; Liu, Gexiu [Department of Physiology, School of Medicine, Jinan University, Guangzhou 510632 (China); Xu, Bin; Lin, Yongliang; Zhang, Peng [Grandhope Biotech Co., Ltd., Building D, #408, Guangzhou International Business Incubator, Guangzhou Science Park, Guangzhou 510663, Guangdong (China); Wei, Xing, E-mail: wei70@hotmail.com [Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Key Lab for Genetic Medicine of Guangdong Province, Jinan University, Guangzhou 510632 (China)

2014-11-01

Current therapy for skin wound healing still relies on skin transplantation. Many studies were done to try to find out ways to replace skin transplantation, but there is still no effective alternative therapy. In this study, decellularized scaffolds were prepared from pig peritoneum by a series of physical and chemical treatments, and scaffolds loaded with hyaluronic acid (HA) and epidermal growth factor (EGF) were tested for their effect on wound healing. MTT assay showed that EGF increased NIH3T3 cell viability and confirmed that EGF used in this study was biologically active in vitro. Scanning electron microscope (SEM) showed that HA stably attached to scaffolds even after soaking in PBS for 48 h. ELISA assay showed that HA increased the adsorption of EGF to scaffolds and sustained the release of EGF from scaffolds. Animal study showed that the wounds covered with scaffolds containing HA and EGF recovered best among all 4 groups and had wound healing rates of 49.86%, 70.94% and 87.41% respectively for days 10, 15 and 20 post-surgery compared to scaffolds alone with wound healing rates of 29.26%, 42.80% and 70.14%. In addition, the wounds covered with scaffolds containing EGF alone were smaller than no EGF scaffolds on days 10, 15 and 20 post-surgery. Hematoxylin–Eosin (HE) staining confirmed these results by showing that on days 10, 15 and 20 post-surgery, the thicker epidermis and dermis layers were observed in the wounds covered with scaffolds containing HA and EGF than scaffolds alone. In addition, the thicker epidermis and dermis layers were also observed in the wounds covered with scaffolds containing EGF than scaffolds alone. Skin appendages were observed on day 20 only in the wound covered with scaffolds containing HA and EGF. These results demonstrate that the scaffolds containing HA and EGF can enhance wound healing. - Highlights: • HA can increase the adsorption of EGF to decellularized scaffolds. • HA can sustain the release of EGF from

Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications

International Nuclear Information System (INIS)

Liu, Yang; Ren, Li; Wang, Yingjun

2013-01-01

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 × 10 −6 cm 2 /s and 7.97 × 10 −7 cm 2 /s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering. - Highlights: ► Crosslinked collagen–gelatin–hyaluronic acid films were fabricated in this study. ► The film had appropriate physical properties. ► Diffusion coefficient of the film was comparable with the human cornea. ► HCEC viability studies confirmed the biocompatibility of the film.

Wavelength-dependent ultraviolet induction of cyclobutane pyrimidine dimers in the human cornea.

Science.gov (United States)

Mallet, Justin D; Rochette, Patrick J

2013-08-01

Exposition to ultraviolet (UV) light is involved in the initiation and the progression of skin cancer. The genotoxicity of UV light is mainly attributed to the induction of cyclobutane pyrimidine dimers (CPDs), the most abundant DNA damage generated by all UV types (UVA, B and C). The human cornea is also exposed to the harmful UV radiations, but no UV-related neoplasm has been reported in this ocular structure. The probability that a specific DNA damage leads to a mutation and eventually to cellular transformation is influenced by its formation frequency. To shed light on the genotoxic effect of sunlight in the human eye, we have analyzed CPD induction in the cornea and the iris following irradiation of ex vivo human eyes with UVA, B or C. The extent of CPD induction was used to establish the penetrance of the different UV types in the human cornea. We show that UVB- and UVC-induced CPDs are concentrated in the corneal epithelium and do not penetrate deeply beyond this corneal layer. On the other hand, UVA wavelengths penetrate deeper and induce CPDs in the entire cornea and in the first layers of the iris. Taken together, our results are undoubtedly an important step towards better understanding the consequences of UV exposure to the human eye.

Porcine respiratory disease complex: Interaction of vaccination and porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and Mycoplasma hyopneumoniae.

Science.gov (United States)

Chae, Chanhee

2016-06-01

Porcine respiratory disease is a multifactorial and complex disease caused by a combination of infectious pathogens, environmental stressors, differences in production systems, and various management practices; hence the name porcine respiratory disease complex (PRDC) is used. Porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and Mycoplasma hyopneumoniae are considered to be the most important pathogens that cause PRDC. Although interactions among the three major respiratory pathogens are well documented, it is also necessary to understand the interaction between vaccines and the three major respiratory pathogens. PRRSV and M. hyopneumoniae are well known to potentiate PCV2-associated lesions; however, PRRSV and mycoplasmal vaccines can both enhance PCV2 viraemia regardless of the effects of the actual PRRSV or M. hyopneumoniae infection. On the other hand, M. hyopneumoniae potentiates the severity of pneumonia induced by PRRSV, and vaccination against M. hyopneumoniae alone is also able to decrease PRRSV viraemia and PRRSV-induced lung lesions in dually infected pigs. This review focuses on (1) interactions between PCV2, PRRSV, and M. hyopneumoniae; and (2) interactions between vaccines and the three major respiratory pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Computational modeling of drug transport across the in vitro cornea.

Science.gov (United States)

Pak, Joseph; Chen, Z J; Sun, Kay; Przekwas, Andrzej; Walenga, Ross; Fan, Jianghong

2018-01-01

A novel quasi-3D (Q3D) modeling approach was developed to model networks of one dimensional structures like tubes and vessels common in human anatomy such as vascular and lymphatic systems, neural networks, and respiratory airways. Instead of a branching network of the same tissue type, this approach was extended to model an interconnected stack of different corneal tissue layers with membrane junction conditions assigned between the tissues. The multi-laminate structure of the cornea presents a unique barrier design and opportunity for investigation using Q3D modeling. A Q3D model of an in vitro rabbit cornea was created to simulate the drug transport across the cornea, accounting for transcellular and paracellular pathways of passive and convective drug transport as well as physicochemistry of lipophilic partitioning and protein binding. Lipophilic Rhodamine B and hydrophilic fluorescein were used as drug analogs. The model predictions for both hydrophilic and lipophilic tracers were able to match the experimental measurements along with the sharp discontinuities at the epithelium-stroma and stroma-endothelium interfaces. This new modeling approach was successfully applied towards pharmacokinetic modeling for use in topical ophthalmic drug design. Copyright © 2017 Elsevier Ltd. All rights reserved.

The effect of single and repeated UVB radiation on rabbit cornea.

Science.gov (United States)

Fris, Miroslav; Tessem, May-Britt; Cejková, Jitka; Midelfart, Anna

2006-12-01

Cumulative effect of ultraviolet radiation (UVR) is an important aspect of UV corneal damage. The purpose of this study was to apply high resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS 1H NMR) spectroscopy to evaluate the effect of single and repeated UV radiation exposure of the same overall dose on the rabbit cornea. Corneal surfaces of 24 normal rabbit eyes were examined for the effects of UVB exposure (312 nm). In the first group (UVB1), animals were irradiated with a single dose (3.12 J/cm2; 21 min) of UVB radiation. The animals in the second group (UVB2) were irradiated three times for 7 min every other day (dose of 1.04 J/cm2; days 1, 3, 5) to give the same overall dose (3.12 J/cm2). The third group served as an untreated control group. One day after the last irradiation, the animals were sacrificed, and the corneas were removed and frozen. HR-MAS 1H NMR spectra from intact corneas were obtained. Special grouping patterns among the tissue samples and the relative percentage changes in particular metabolite concentrations were evaluated using modern statistical methods (multivariate analysis, one-way ANOVA). The metabolic profile of both groups of UVB-irradiated samples was significantly different from the control corneas. Substantial decreases in taurine, hypo-taurine and choline-derivatives concentrations and substantial elevation in glucose and betaine levels were observed following the UVR exposure. There was no significant difference between the effect of a single and repeated UVB irradiation of the same overall dose. For the first time, the effects of single and repeated UVR doses on the metabolic profile of the rabbit cornea were analysed and compared. The combination of HR-MAS 1H NMR spectroscopy and modern statistical methods (multivariate analysis, one-way ANOVA) proved suitable to assess the overall view of the metabolic alterations in the rabbit corneal tissue following UVB radiation exposure.

Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2

DEFF Research Database (Denmark)

McNair, I.; Marshall, M.; McNeilly, F.

2004-01-01

A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assa...... than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus....

Thermodynamic measurement after cooling the cornea with intact epithelium and lid manipulation.

Science.gov (United States)

de Ortueta, Diego; Magnago, Thomas; Arba-Mosquera, Samuel

2015-01-01

To characterize the rate of change of ocular surface temperature (OST) under lid manipulation after cooling the intact cornea with balanced salt solution (BSS). In a patient for refractive surgery, prior to the ablation, the temperature of the cornea was continuously recorded with a high speed infrared (350Hz) camera. Two millilitre of chilled BSS with a temperature of 8.6°Celsius (°C) was instilled for about 3s. Using exponential functions, the three contributions have been determined, subjacent corneal layers, environment, and chilled BSS. The mean temperature of the cornea preoperatively was 34.5°C. After applying the chilled BSS the temperature decreased about 14°C down to an OST of 20°C and the time needed afterwards to get the normal (OST) temperature of about 30°C was 40s. Due to the inserted speculum and missing blink, OST did not reach the original OST of 34.5°C and faded at about 32.5°C. According to our best fitted model, absolute value of each contributing component was 31.4°C (subjacent corneal layers), 26.8°C (environment) and 8.6°C (BSS). Applying chilled BSS to the cornea quickly reduces the temperature of the cornea with a thermal relaxation time of 3s and a amplitude decrease of 8.6°C. This together with a relaxation time of 7s for subjacent corneal layers, and 184s for environment after instillation of BSS combined with a well-controlled environment provides a period of 40s of corneal temperature below baseline, which may be of clinical benefit when applying chilled BSS immediately before or immediately after ablation. Copyright © 2014 Spanish General Council of Optometry. Published by Elsevier Espana. All rights reserved.

[Results of an internet-based survey amongst members of the German Ophthalmological Society concerning postmortem cornea donation].

Science.gov (United States)

Uhlig, C E; Promesberger, J; Hirschfeld, G; Koch, R; Reinhard, T; Seitz, B

2012-12-01

Analysis of willingness for postmortem cornea donation by professionals in ophthalmology and their motives in favor of or against donation. 3887 members of the German Ophthalmological Society received an anonymous questionnaire concerning sociodemographic background, physical health, experiences with organ explantation and their former engagement and motives concerning organ and cornea donation. 722 of the questionnaires were partially and 533 completely answered with an average willingness for cornea donation of 79.4%. Significant parameters for cornea donation were gender, former experience with organ explantation, ophthalmological health and fear of false diagnosis of brain death, worse medical treatment or organ commercialization. Of the participants 53.9% suggested the internet as a favorite source of information in this matter. The factors which had a significant impact on cornea donation in this survey seem to be mainly a result of insufficient information. Detailed information regarding this topic should preferentially be presented on internet pages of professional societies and could probably increase donation approval of DOG members.

Crosslinked collagen-gelatin-hyaluronic acid biomimetic film for cornea tissue engineering applications

Energy Technology Data Exchange (ETDEWEB)

Liu, Yang; Ren, Li, E-mail: psliren@scut.edu.cn; Wang, Yingjun, E-mail: imwangyj@163.com

2013-01-01

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)-gelatin (Gel)-hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA = 6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43 Multiplication-Sign 10{sup -6} cm{sup 2}/s and 7.97 Multiplication-Sign 10{sup -7} cm{sup 2}/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering. - Highlights: Black-Right-Pointing-Pointer Crosslinked collagen-gelatin-hyaluronic acid films were fabricated in this study. Black-Right-Pointing-Pointer The film had appropriate physical properties. Black-Right-Pointing-Pointer Diffusion coefficient of the film was comparable with the human cornea. Black-Right-Pointing-Pointer HCEC viability studies confirmed the biocompatibility of the film.

Mesothelial proteins are expressed in the human cornea

Czech Academy of Sciences Publication Activity Database

Jirsová, K.; Neuwirth, Aleš; Kalašová, S.; Veselá, V.; Merjava, S.

2010-01-01

Roč. 91, č. 5 (2010), s. 623-629 ISSN 0014-4835 Institutional research plan: CEZ:AV0Z50520514 Keywords : cornea * HBME-1 (Hector Battifora mesothelial cell-1) protein * calbindin 2 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.817, year: 2010

Repair of facial nerve defects with decellularized artery allografts containing autologous adipose-derived stem cells in a rat model.

Science.gov (United States)

Sun, Fei; Zhou, Ke; Mi, Wen-Juan; Qiu, Jian-Hua

2011-07-20

The purpose of this study was to investigate the effects of a decellularized artery allograft containing autologous adipose-derived stem cells (ADSCs) on an 8-mm facial nerve branch lesion in a rat model. At 8 weeks postoperatively, functional evaluation of unilateral vibrissae movements, morphological analysis of regenerated nerve segments and retrograde labeling of facial motoneurons were all analyzed. Better regenerative outcomes associated with functional improvement, great axonal growth, and improved target reinnervation were achieved in the artery-ADSCs group (2), whereas the cut nerves sutured with artery conduits alone (group 1) achieved inferior restoration. Furthermore, transected nerves repaired with nerve autografts (group 3) resulted in significant recovery of whisking, maturation of myelinated fibers and increased number of labeled facial neurons, and the latter two parameters were significantly different from those of group 2. Collectively, though our combined use of a decellularized artery allograft with autologous ADSCs achieved regenerative outcomes inferior to a nerve autograft, it certainly showed a beneficial effect on promoting nerve regeneration and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

Science.gov (United States)

Kaji, Yuichi; Akiyama, Toshihiro; Segawa, Hiroki; Oshika, Tetsuro; Kano, Hideaki

2017-11-01

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

Xanthelasmata, arcus corneae, and ischaemic vascular disease and death in general population: prospective cohort study

DEFF Research Database (Denmark)

Christoffersen, Mette; Frikke-Schmidt, Ruth; Schnohr, Peter

2011-01-01

To test the hypothesis that xanthelasmata and arcus corneae, individually and combined, predict risk of ischaemic vascular disease and death in the general population.......To test the hypothesis that xanthelasmata and arcus corneae, individually and combined, predict risk of ischaemic vascular disease and death in the general population....

Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging

DEFF Research Database (Denmark)

Karring, Henrik; Runager, Kasper; Valnickova, Zuzana

2010-01-01

Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging...... trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea...... and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing...

An active artificial cornea with the function of inducing new corneal tissue generation in vivo-a new approach to corneal tissue engineering

International Nuclear Information System (INIS)

Huang Yaoxiong; Li Qinhua

2007-01-01

An active artificial cornea which can perform the function of inducing new cornea generation in vivo but does not need culture cells in vitro and which has similar optical and mechanical properties to those of the human cornea was constructed. An animal keratoplasty experiment using the artificial cornea as the implant showed that the animals' corneas could keep smooth surface and clear stroma postoperatively, and that the repopulation of the host's keratocytes, the degradation of the implant and new corneal tissue generation were completed at 5-6 months after surgery. Such an artificial cornea has several advantages over other corneal equivalents constructed in the typical way of tissue engineering: in having similar mechanical and optical properties to those of the human cornea and with no exogenetic cells, it can be used universally in different implantation surgeries without immunoreaction; it is easy to prepare and process into different shapes and sizes on a large scale, and suitable for long-distance transportation and long-term storage. All these characteristics make it a new approach to cornea tissue engineering having potential in many clinical applications

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

Science.gov (United States)

Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2016-05-01

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

Effects of genipin corneal crosslinking in rabbit corneas.

Science.gov (United States)

Avila, Marcel Y; Narvaez, Mauricio; Castañeda, Juan P

2016-07-01

To evaluate the effect of genipin, a natural crosslinking agent, in rabbit eyes. Department of Ophthalmology, Universidad Nacional de Colombia Centro de Tecnologia Oftalmica, Bogotá, Colombia. Experimental study. Ex vivo rabbit eyes (16; 8 rabbits) were treated with genipin 1.00%, 0.50%, and 0.25% for 5 minutes with a vacuum device to increase corneal permeability. Penetration was evaluated using Scheimpflug pachymetry (Pentacam). In the in vivo model (20 rabbits; 1 eye treated, 1 eye with vehicle), corneas were crosslinked with genipin as described. Corneal curvature, corneal pachymetry, and intraocular pressure (IOP) assessments as well as slitlamp examinations were performed 0, 7, 30, and 60 days after treatment. In the ex vivo model, Scheimpflug pachymetry showed deep penetration in the rabbit corneas with an increase in corneal density and a dose-dependent relationship. Corneal flattening was observed in treated eyes (mean 4.4 diopters ± 0.5 [SD]) compared with the control eyes. Pachymetry and IOP were stable in all evaluations. No eye showed toxicity in the anterior chamber or in the lens. Corneal crosslinking induced by genipin produced significant flattening of the cornea with no toxicity in rabbit eyes. This crosslinking could be useful in the treatment of corneal ectasia and in the modification of corneal curvature. None of the authors has a financial or proprietary interest in any material or method mentioned. Copyright © 2016 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Novel decellularized liver matrix-alginate hybrid gel beads for the 3D culture of hepatocellular carcinoma cells.

Science.gov (United States)

Sun, Dongsheng; Liu, Yang; Wang, Huihui; Deng, Fei; Zhang, Ying; Zhao, Shan; Ma, Xiaojun; Wu, Huijian; Sun, Guangwei

2018-04-01

Developing reliable three-dimensional (3D) cell culture systems that can mimic native tumor microenvironments is necessary for investigating the mechanism of hepatocellular carcinoma (HCC) metastasis and screen therapeutic drugs. In the present study, we developed decellularized liver matrix-alginate (DLM-ALG) hybrid gel beads. DLM powder was prepared by optimized decellularization methods and liquid nitrogen grinding. DLM-ALG beads were generated by dropping alginate solution containing DLM powder into a gelling bath. DLM powder concentration in alginate solution was ≤1% (w/v) and had no effect on the sphericity and mechanical stability of the beads. In addition, HCCLM3 cells cultured in 1% (w/v) DLM-ALG beads presented gradually enhanced viability during in vitro culture. The protein expression of urokinase plasminogen activator system and activity of matrix metalloproteinases (MMPs) of HCCLM3 cells, including MMP2 and MMP9, were more significantly promoted in DLM-ALG beads compared with that in conventional ALG beads without DLM powder. Moreover, the dose-dependent increase in HCCLM3 cell MMP activities was observed along with the DLM powder concentration in 0.5% and 1% DLM-ALG groups. Therefore, DLM-ALG beads might serve as a novel 3D culture system for exploring the mechanisms of HCC metastasis and screening therapeutic drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Therapeutic effect of para aminobenzoic acid (PABA) on the rat cornea administered in different ways after x-irradiation

International Nuclear Information System (INIS)

Panova, I.G.; Mel'nikova, I.I.; Stroeva, O.G.

1997-01-01

Comparative analysis of cytological state of nuclear apparatus in the mice cornea basal layer is carried out by two various method of introducing the para-aminobenzoic acid (PABA) after x-ray irradiation: 1) subcutaneous injections and 2) application of the solution on the cornea. The fact that PABA produced medicinal effect on the cornea of the irradiated mice by subcutaneous injections, shows that this action proceeds on the whole-body level

Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

Science.gov (United States)

Zhang, Ying; Li, Jingting; Davis, Mary E.; Pei, Ming

2015-01-01

As a tissue-specific stem cell for chondrogenesis, synovium-derived stem cells (SDSCs) are a promising cell source for cartilage repair. However, a small biopsy can only provide a limited number of cells. Cell senescence from both in vitro expansion and donor age presents a big challenge for stem cell based cartilage regeneration. Here we found that expansion on decellularized extracellular matrix (dECM) full of three-dimensional nanostructured fibers provided SDSCs with unique surface profiles, low elasticity but large volume as well as fibroblast-like shape. dECM expanded SDSCs yielded larger pellets with intensive staining of type II collagen and sulfated glycosaminoglycans compared to those grown on plastic flasks while SDSCs grown in ECM yielded 28-day pellets with minimal matrix as evidenced by pellet size and chondrogenic marker staining, which was confirmed by both biochemical data and real-time PCR data. Our results also found lower levels of inflammatory genes in dECM expanded SDSCs that might be responsible for enhanced chondrogenic differentiation. Despite an increase in type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Wnt and MAPK signals were actively involved in both expansion and chondrogenic induction of dECM expanded cells. Since young and healthy people can be potential donors for this matrix expansion system and decellularization can minimize immune concerns, human SDSCs expanded on this future commercially available dECM could be a potential cell source for autologous cartilage repair. PMID:25861949

Solid organ fabrication: comparison of decellularization to 3D bioprinting.

Science.gov (United States)

Jung, Jangwook P; Bhuiyan, Didarul B; Ogle, Brenda M

2016-01-01

Solid organ fabrication is an ultimate goal of Regenerative Medicine. Since the introduction of Tissue Engineering in 1993, functional biomaterials, stem cells, tunable microenvironments, and high-resolution imaging technologies have significantly advanced efforts to regenerate in vitro culture or tissue platforms. Relatively simple flat or tubular organs are already in (pre)clinical trials and a few commercial products are in market. The road to more complex, high demand, solid organs including heart, kidney and lung will require substantive technical advancement. Here, we consider two emerging technologies for solid organ fabrication. One is decellularization of cadaveric organs followed by repopulation with terminally differentiated or progenitor cells. The other is 3D bioprinting to deposit cell-laden bio-inks to attain complex tissue architecture. We reviewed the development and evolution of the two technologies and evaluated relative strengths needed to produce solid organs, with special emphasis on the heart and other tissues of the cardiovascular system.

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

Science.gov (United States)

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-06-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

Property-based design: optimization and characterization of polyvinyl alcohol (PVA) hydrogel and PVA-matrix composite for artificial cornea.

Science.gov (United States)

Jiang, Hong; Zuo, Yi; Zhang, Li; Li, Jidong; Zhang, Aiming; Li, Yubao; Yang, Xiaochao

2014-03-01

Each approach for artificial cornea design is toward the same goal: to develop a material that best mimics the important properties of natural cornea. Accordingly, the selection and optimization of corneal substitute should be based on their physicochemical properties. In this study, three types of polyvinyl alcohol (PVA) hydrogels with different polymerization degree (PVA1799, PVA2499 and PVA2699) were prepared by freeze-thawing techniques. After characterization in terms of transparency, water content, water contact angle, mechanical property, root-mean-square roughness and protein adsorption behavior, the optimized PVA2499 hydrogel with similar properties of natural cornea was selected as a matrix material for artificial cornea. Based on this, a biomimetic artificial cornea was fabricated with core-and-skirt structure: a transparent PVA hydrogel core, surrounding by a ringed PVA-matrix composite skirt that composed of graphite, Fe-doped nano hydroxyapatite (n-Fe-HA) and PVA hydrogel. Different ratio of graphite/n-Fe-HA can tune the skirt color from dark brown to light brown, which well simulates the iris color of Oriental eyes. Moreover, morphologic and mechanical examination showed that an integrated core-and-skirt artificial cornea was formed from an interpenetrating polymer network, no phase separation appeared on the interface between the core and the skirt.

Multiparameter thermo-mechanical OCT-based characterization of laser-induced cornea reshaping

Science.gov (United States)

Zaitsev, Vladimir Yu.; Matveyev, Alexandr L.; Matveev, Lev A.; Gelikonov, Grigory V.; Vitkin, Alex; Omelchenko, Alexander I.; Baum, Olga I.; Shabanov, Dmitry V.; Sovetsky, Alexander A.; Sobol, Emil N.

2017-02-01

Phase-sensitive optical coherence tomography (OCT) is used for visualizing dynamic and cumulative strains and corneashape changes during laser-produced tissue heating. Such non-destructive (non-ablative) cornea reshaping can be used as a basis of emerging technologies of laser vision correction. In experiments with cartilaginous samples, polyacrilamide phantoms and excised rabbit eyes we demonstrate ability of the developed OCT system to simultaneously characterize transient and cumulated strain distributions, surface displacements, scattering tissue properties and possibility of temperature estimation via thermal-expansion measurements. The proposed approach can be implemented in perspective real-time OCT systems for ensuring safety of new methods of laser reshaping of cornea.

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

International Nuclear Information System (INIS)

Cenedella, R.J.; Fleschner, C.R.

1989-01-01

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3 H 2 O and [ 14 C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14 C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3 H of 3 H 2 O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered

Air-pulse OCE for assessment of age-related changes in mouse cornea in vivo

International Nuclear Information System (INIS)

Li, Jiasong; Wang, Shang; Singh, M; Larin, K V; Aglyamov, S; Emelianov, S; Twa, M D

2014-01-01

We demonstrate the use of phase-stabilized swept source optical coherence elastography (PhS-SSOCE) to assess the relaxation rate of deformation created by a focused air-pulse in tissue-mimicking gelatin phantoms of various concentrations and mouse corneas of different ages in vivo. The results show that the relaxation rate can be quantified and is different for gels with varying concentrations of gelatin and mouse corneas of different ages. The results indicate that gel phantoms with higher concentrations of gelatin as well as older mouse corneas have faster relaxation rates indicating stiffer material. This non-contact and non-invasive measurement technique utilizes low surface displacement amplitude (in µm scale) for tissue excitation and, therefore, can be potentially used to study the biomechanical properties of ocular and other sensitive tissues. (letter)

Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux.

Science.gov (United States)

Karla, Pradeep K; Quinn, Tim L; Herndon, Betty L; Thomas, Priscilla; Pal, Dhananjay; Mitra, Ashim

2009-04-01

The purpose of this manuscript is to investigate the presence of nucleoside/nucleotide efflux transporter in cornea and to evaluate the role in ocular drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis and immunostaining were employed to establish molecular presence of multidrug resistance associated protein 5 (MRP5) on cornea. Corneal efflux by MRP5 was studied with bis(POM)-PMEA and acyclovir using rabbit and human corneal epithelial cells along with MRP5 over expressing cells (MDCKII-MRP5). Ex vivo studies using excised rabbit cornea and in vivo ocular microdialysis in male New Zealand white rabbits were used to further evaluate the role of MRP5 in conferring ocular drug resistance. RT-PCR confirms the expression of MRP5 in both rabbit and human corneal epithelial cells along with MDCKII-MRP5 cells. Immunoprecipitation followed by Western blot analysis using a rat (M511-54) monoclonal antibody that reacts with human epitope confirms the expression of MRP5 protein in human corneal epithelial cells and MDCKII-MRP5 cells. Immunostaining performed on human cornea indicates the localization of this efflux pump on both epithelium and endothelium. Efflux studies reveal that depletion of ATP decreased PMEA efflux significantly. MRP5 inhibitors also diminished PMEA and acyclovir efflux. However, depletion of glutathione did not alter efflux. MDR1 and MRP2 did not contribute to PMEA efflux. However, MRP2 is involved in acyclovir efflux while MDR1 do not participate in this process. TLC/autoradiography suggested the conversion of bis(POM)-PMEA to PMEA in rabbit and human corneal epithelial cells. Two well known antiglaucoma drugs, bimatoprost and latanoprost were rapidly effluxed by MRP5. Ex vivo study on intact rabbit corneas demonstrated accumulation of PMEA in cornea in the presence of ATP-depleting medium. In vivo ocular pharmacokinetics also revealed a significant increase in maximum aqueous humor concentration (C(max)) and area under the

Optics of the human cornea influence the accuracy of stereo eye-tracking methods: a simulation study

NARCIS (Netherlands)

Barsingerhorn, A.D.; Boonstra, F.N.; Goossens, H.H.L.M.

2017-01-01

Current stereo eye-tracking methods model the cornea as a sphere with one refractive surface. However, the human cornea is slightly aspheric and has two refractive surfaces. Here we used ray-tracing and the Navarro eye-model to study how these optical properties affect the accuracy of different

Effect of glaucoma tube shunt parameters on cornea endothelial cells in patients with Ahmed valve implants.

Science.gov (United States)

Koo, Euna B; Hou, Jing; Han, Ying; Keenan, Jeremy D; Stamper, Robert L; Jeng, Bennie H

2015-01-01

The aim of this study was to assess the effect of various tube parameters on corneal endothelial cell density (ECD) after insertion of Ahmed valves. Thirty-nine eyes of 33 patients with previous superotemporal (ST) Ahmed valve implantation and 20 eyes of 13 participants with previous uncomplicated phacoemulsification and intraocular lens implantation but no history of glaucoma surgery were evaluated. Various tube parameters were measured with anterior segment optical coherence tomography. ST, central, and inferonasal (IN) ECD and pachymetry were measured. Endothelial cell loss and corneal thickness in the ST cornea was compared with those in the IN cornea. The mean age of the operated patients was 58 ± 22 years, and the mean time since glaucoma surgery was 2.5 ± 2.6 years. Thirty-two of the 39 study eyes were pseudophakic. The ECD was significantly lower in the ST endothelium than in the IN endothelium in eyes with glaucoma tube surgery (P < 0.001), although this relative reduction in ST ECD was not greater than that seen in pseudophakic control eyes (P = 0.16). In univariate analysis, tube angle relative to the cornea and distance from the tip of the tube to the cornea were significant risk factors for decreased ST endothelial cell loss when assessed relative to the IN ECD (P = 0.01 and P = 0.02, respectively). In multivariate analysis, only the distance of the tube tip to the cornea remained significantly associated with ST endothelial cell loss. Although this was a retrospective study with inherent limitations, tubes that are closer to the cornea seem to lead to increased loss of adjacent endothelial cells.

Tracing the fate of limbal epithelial progenitor cells in the murine cornea.

Science.gov (United States)

Di Girolamo, N; Bobba, S; Raviraj, V; Delic, N C; Slapetova, I; Nicovich, P R; Halliday, G M; Wakefield, D; Whan, R; Lyons, J G

2015-01-01

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation. © 2014 AlphaMed Press.

Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas.

Directory of Open Access Journals (Sweden)

Umberto Rosani

Full Text Available Atmospheric pressure cold plasma (APCP might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS that kill microorganisms without substantially affecting human cells.In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC was able to inhibit or prevent damage and cell death.Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds. Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h.These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.

Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

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Pati, Falguni; Cho, Dong-Woo

2017-01-01

Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

Optics of the human cornea influence the accuracy of stereo eye-tracking methods: a simulation study.

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Barsingerhorn, A D; Boonstra, F N; Goossens, H H L M

2017-02-01

Current stereo eye-tracking methods model the cornea as a sphere with one refractive surface. However, the human cornea is slightly aspheric and has two refractive surfaces. Here we used ray-tracing and the Navarro eye-model to study how these optical properties affect the accuracy of different stereo eye-tracking methods. We found that pupil size, gaze direction and head position all influence the reconstruction of gaze. Resulting errors range between ± 1.0 degrees at best. This shows that stereo eye-tracking may be an option if reliable calibration is not possible, but the applied eye-model should account for the actual optics of the cornea.

Optical properties of an anterior lamellar human cornea model based on fibrin-agarose

Science.gov (United States)

Ionescu, Ana M.; Cardona, Juan de la Cruz; Ghinea, Razvan; Garzón, Ingrid; González-Andrades, Miguel; Alaminos, Miguel; Pérez, Maria del Mar

2017-08-01

The optical evaluation carried out using the Inverse Adding-Doubling (IAD) method to determine the scattering and the absorption coefficients of the bioengineered human corneal stromas showed that this type of artificial biomaterials shared many similarities with native control cornea after four weeks of development in culture. Their absorption and reduced scattering coefficients values were higher than the ones of the control cornea, but their spectral behaviors of both coefficients were similar. Time of development in culture was an influencing factor on the results.

7 CFR 1230.18 - Porcine animal.

Science.gov (United States)

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.18 Section 1230.18 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... animal. Porcine animal means a swine, that is raised as (a) a feeder pig, that is, a young pig sold to...

Perfusion decellularization of a human limb: A novel platform for composite tissue engineering and reconstructive surgery.

Directory of Open Access Journals (Sweden)

Mattia Francesco Maria Gerli

Full Text Available Muscle and fasciocutaneous flaps taken from autologous donor sites are currently the most utilized approach for trauma repair, accounting annually for 4.5 million procedures in the US alone. However, the donor tissue size is limited and the complications related to these surgical techniques lead to morbidities, often involving the donor sites. Alternatively, recent reports indicated that extracellular matrix (ECM scaffolds boost the regenerative potential of the injured site, as shown in a small cohort of volumetric muscle loss patients. Perfusion decellularization is a bioengineering technology that allows the generation of clinical-scale ECM scaffolds with preserved complex architecture and with an intact vascular template, from a variety of donor organs and tissues. We recently reported that this technology is amenable to generate full composite tissue scaffolds from rat and non-human primate limbs. Translating this platform to human extremities could substantially benefit soft tissue and volumetric muscle loss patients providing tissue- and species-specific grafts. In this proof-of-concept study, we show the successful generation a large-scale, acellular composite tissue scaffold from a full cadaveric human upper extremity. This construct retained its morphological architecture and perfusable vascular conduits. Histological and biochemical validation confirmed the successful removal of nuclear and cellular components, and highlighted the preservation of the native extracellular matrix components. Our results indicate that perfusion decellularization can be applied to produce human composite tissue acellular scaffolds. With its preserved structure and vascular template, these biocompatible constructs, could have significant advantages over the currently implanted matrices by means of nutrient distribution, size-scalability and immunological response.

Collagen organization in the chicken cornea and structural alterations in the retinopathy, globe enlarged (rge) phenotype--an X-ray diffraction study.

Science.gov (United States)

Boote, Craig; Hayes, Sally; Jones, Simon; Quantock, Andrew J; Hocking, Paul M; Inglehearn, Chris F; Ali, Manir; Meek, Keith M

2008-01-01

An investigation into the collagenous structure of the mature avian cornea is presented. Wide-angle X-ray diffraction is employed to assess collagen organization in 9-month-old chicken corneas. The central 2-4mm corneal region features a preponderance of fibrils directed along the superior-inferior and nasal-temporal orthogonal meridians. More peripherally the orientation of fibrils alters in favor of a predominantly tangential arrangement. The chicken cornea appears to be circumscribed by an annulus of fibrils that extends into the limbus. The natural arrangement of collagen in the chicken cornea is discussed in relation to corneal shape and the mechanical requirements of avian corneal accommodation. Equivalent data are also presented from age-matched blind chickens affected with the retinopathy, globe enlarged (rge) mutation, characterized by an abnormally thick and flat cornea. The data indicate considerable realignment and redistribution of collagen lamellae in the peripheral rge cornea. In contrast to normal chickens, no obvious tangential collagen alignment was evident in the periphery of rge corneas. In mammals, the presence of a limbal fibril annulus is believed to be important in corneal shape preservation. We postulate that corneal flattening in rge chickens may be related to biomechanical changes brought about by an alteration in collagen arrangement at the corneal periphery.

Clinical Evaluation of Decellularized Nerve Allograft with Autologous Bone Marrow Stem Cells to Improve Peripheral Nerve Repair and Functional Outcomes

Science.gov (United States)

2017-07-01

with autologous mesenchymal stem cells . Exp Neurol. 2007 Apr; 204(2):658-66. 19. Dezawa M., et al., Sciatic nerve regeneration in rats induced by...36 23. Mimura T., et al., Peripheral nerve regeneration by transplantation of bone marrow stromal cell -derived Schwann cells in adult rats. J...AWARD NUMBER: W81XWH-15-2-0026 TITLE: Clinical Evaluation of Decellularized Nerve Allograft with Autologous Bone Marrow Stem Cells to Improve

Computer driven optical keratometer and method of evaluating the shape of the cornea

Science.gov (United States)

Baroth, Edmund C. (Inventor); Mouneimme, Samih A. (Inventor)

1994-01-01

An apparatus and method for measuring the shape of the cornea utilize only one reticle to generate a pattern of rings projected onto the surface of a subject's eye. The reflected pattern is focused onto an imaging device such as a video camera and a computer compares the reflected pattern with a reference pattern stored in the computer's memory. The differences between the reflected and stored patterns are used to calculate the deformation of the cornea which may be useful for pre-and post-operative evaluation of the eye by surgeons.

[Riboflavin photoprotection with cross-linking effect in photorefractive ablation of the cornea].

Science.gov (United States)

Kornilovskiy, I M; Sultanova, A I; Burtsev, A A

2016-01-01

Photorefractive ablation is inevitably accompanied by oxidative stress of the cornea and weakening of its biomechanical and photoprotective properties. To validate the expediency of riboflavin use in photorefractive ablation for photoprotection of the cornea and cross-linking. The effects of riboflavin use in photorefractive ablation was first studied in a series of in vitro and in vivo experiments performed on 56 eyes of 28 rabbits, and then on 232 eyes of 142 patients with different degrees of myopia. Biomechanical testing of corneal samples was performed with Zwick/RoellВZ 2.5/TN1S tensile-testing machine. Transepithelial photorefractive keratectomy (TransPRK) and femtosecond laser-assisted in situ keratomileusis (Femto-LASIK) were performed on Wavelight-Allegretto200, MEL-80, and WaveLight-EX500 excimer laser systems and also VisuMax and WaveLight-FS200 femtosecond lasers. For preliminary examinations, an appropriate set of diagnostic tools was used. In vivo experiments have proved that, in the absence of conservative therapy, riboflavin is able to produce both photoprotective and cross-linking effects to the cornea. Corneal syndrome was thus reduced and re-epithelialization after TransPRK accelerated. Biomechanical testing of corneal samples revealed an increase in tolerated load from 12.9±1.4 N to 18.3±1.2 N (p=0.0002) and tensile strength from 8.6±1.7 MPa to 12.4±1.7 MPa (p=0.007). Clinical studies conducted in a group of patients with mild to moderate myopia have also confirmed the photoprotective effect of riboflavin at months 1-12 after TransPRK. There were no significant differences in uncorrected visual acuity (ranged from 0.80±0.16 to 0.85±0.15) and corrected visual acuity at baseline (0.83±0.14). Evaluation of the optical and refractive effect achieved after Femto-LASIK with riboflavin photoprotection in the fellow eye has shown that this technique is not inferior to the traditional one as to its refractive accuracy, but provides better

Intra-Ocular Pressure Measurement in a Patient with a Thin, Thick or Abnormal Cornea.

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Clement, Colin I; Parker, Douglas G A; Goldberg, Ivan

2016-01-01

Accurate measurement of intra-ocular pressure is a fundamental component of the ocular examination. The most common method of measuring IOP is by Goldmann applanation tonometry, the accuracy of which is influenced by the thickness and biomechanical properties of the cornea. Algorithms devised to correct for corneal thickness to estimate IOP oversimplify the effects of corneal biomechanics. The viscous and elastic properties of the cornea influence IOP measurements in unpredictable ways, a finding borne out in studies of patients with inherently abnormal and surgically altered corneal biomechanics. Dynamic contour tonometry, rebound tonometry and the ocular response analyzer provide useful alternatives to GAT in patients with abnormal corneas, such as those who have undergone laser vision correction or keratoplasty. This article reviews the various methods of intra-ocular pressure measurement available to the clinician and the ways in which their utility is influenced by variations in corneal thickness and biomechanics.

Gastrin-releasing peptide in the porcine pancreas

DEFF Research Database (Denmark)

Holst, J J; Poulsen, Steen Seier

1987-01-01

to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during...... electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas...... in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact...

Excimer laser ablation of the cornea

Science.gov (United States)

Pettit, George H.; Ediger, Marwood N.; Weiblinger, Richard P.

1995-03-01

Pulsed ultraviolet laser ablation is being extensively investigated clinically to reshape the optical surface of the eye and correct vision defects. Current knowledge of the laser/tissue interaction and the present state of the clinical evaluation are reviewed. In addition, the principal findings of internal Food and Drug Administration research are described in some detail, including a risk assessment of the laser-induced-fluorescence and measurement of the nonlinear optical properties of cornea during the intense UV irradiation. Finally, a survey is presented of the alternative laser technologies being explored for this ophthalmic application.

Low-dose radiation effects on the evolution of chronic dystrophical processes in cornea and clouding of crystalline lens

International Nuclear Information System (INIS)

Gajdaj, Yu.V.; Gajdaj, V.M.

1993-01-01

Low-dose radiation effects on the course of chronic dystrophical processes in cornea and the dynamics of crystalline lens clouding of involution age genesis are investigated in the patients participated in Chernobyl accident response. Examples of the concrete pathological cases are considered. It was stated that the above dose effects led to exacerbation of the chronic slack dystrophical processes in cornea and intensification of the development of cornea primary dystrophy. In a number of cases the intensification of development of crystalline lens clouding takes place resulted in the cataract for 2-3 years

Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells.

Science.gov (United States)

Ji, Chun-Miao; Wang, Bin; Zhou, Jiyong; Huang, Yao-Wei

2018-04-01

A monkey cell line Vero (ATCC CCL-81) is commonly used for porcine epidemic diarrhea virus (PEDV) propagation in vitro. However, it is still controversial whether the porcine aminopeptidase N (pAPN) counterpart on Vero cells (Vero-APN) confers PEDV entry. We found that endogenous expression of Vero-APN was undetectable in the mRNA and the protein levels in Vero cells. We cloned the partial Vero-APN gene (3340-bp) containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach. PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment. A Vero cells stably expressing pAPN did not increase PEDV production. SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection. The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry. Copyright © 2018 Elsevier Inc. All rights reserved.

Colocalization of Galectin-3 With CD147 Is Associated With Increased Gelatinolytic Activity in Ulcerating Human Corneas.

Science.gov (United States)

Cruzat, Andrea; Gonzalez-Andrades, Miguel; Mauris, Jérôme; AbuSamra, Dina B; Chidambaram, Preethi; Kenyon, Kenneth R; Chodosh, James; Dohlman, Claes H; Argüeso, Pablo

2018-01-01

Galectin-3 is a carbohydrate-binding protein known to promote expression of matrix metalloproteinases, a hallmark of ulceration, through interaction with the extracellular matrix metalloproteinase inducer CD147. The aim of this study was to investigate the distribution of galectin-3 in corneas of patients with ulcerative keratitis and to determine its relationship to CD147 and the presence of gelatinolytic activity. This was an observational case series involving donor tissue from 13 patients with active corneal ulceration and 6 control corneas. Fixed-frozen sections of the corneas were processed to localize galectin-3 and CD147 by immunofluorescence microscopy. Gelatinolytic activity was detected by in situ zymography. Tissue from patients with active corneal ulceration showed a greater galectin-3 immunoreactivity in basal epithelia and stroma compared with controls. Immunofluorescence grading scores revealed increased colocalization of galectin-3 and CD147 in corneal ulcers at the epithelial-stromal junction and within fibroblasts. Quantitative analysis using the Manders' colocalization coefficient demonstrated significant overlap in corneas from patients with ulcerative keratitis (M1 = 0.29; M2 = 0.22) as opposed to control corneas (M1 = 0.01, P < 0.01; M2 = 0.02, P < 0.05). In these experiments, there was a significant positive correlation between the degree of galectin-3 and CD147 colocalization and the presence of gelatinolytic activity. Our results indicate that concomitant stimulation and colocalization of galectin-3 with CD147 are associated with increased gelatinolytic activity in the actively ulcerating human cornea and suggest a mechanism by which galectin-3 may contribute to the degradation of extracellular matrix proteins during ulceration.

Quantification of Porcine Vocal Fold Geometry.

Science.gov (United States)

Stevens, Kimberly A; Thomson, Scott L; Jetté, Marie E; Thibeault, Susan L

2016-07-01

The aim of this study was to quantify porcine vocal fold medial surface geometry and three-dimensional geometric distortion induced by freezing the larynx, especially in the region of the vocal folds. The medial surface geometries of five excised porcine larynges were quantified and reported. Five porcine larynges were imaged in a micro-CT scanner, frozen, and rescanned. Segmentations and three-dimensional reconstructions were used to quantify and characterize geometric features. Comparisons were made with geometry data previously obtained using canine and human vocal folds as well as geometries of selected synthetic vocal fold models. Freezing induced an overall expansion of approximately 5% in the transverse plane and comparable levels of nonuniform distortion in sagittal and coronal planes. The medial surface of the porcine vocal folds was found to compare reasonably well with other geometries, although the compared geometries exhibited a notable discrepancy with one set of published human female vocal fold geometry. Porcine vocal folds are qualitatively geometrically similar to data available for canine and human vocal folds, as well as commonly used models. Freezing of tissue in the larynx causes distortion of around 5%. The data can provide direction in estimating uncertainty due to bulk distortion of tissue caused by freezing, as well as quantitative geometric data that can be directly used in developing vocal fold models. Copyright © 2016 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium

Directory of Open Access Journals (Sweden)

Yun Wang

2013-10-01

Full Text Available AIM: To observe the clinical effectiveness and practicality the autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium.METHODS: Of the 53 recurrent pterygium patients(57 eyes, after all pathological tissues were removed, underwent the autologous conjunctiva transplantation with stem cells on edge of cornea which were locked above conjunctival transplantation of the operated eye.RESULTS: Postopretive follow-up was 1-12 months for all 57 eyes, of which 3 eyes(5%relapsed. The corneoscleral autolysis was occurred in one eye and surgery treatment was conducted. Corneal wounds were healing and transplantations survived well for the remaining 53 patients without obvious surgical marks. Cure rate was 93%.CONCLUSION: Autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium can meet the aesthetic requirements of the some patients, with the advantages of obtaining material easily, faster wound healing, lower postoperative recurrence rate, meeting the aesthetic needs of some patients and improving postoperative results. Thus, it is an ideal surgery and is worthy of applying on primary hospital.

The Naïve Murine Cornea as a Model System to Identify Novel Endogenous Regulators of Lymphangiogenesis: TRAIL and rtPA.

Science.gov (United States)

Regenfuß, Birgit; Dreisow, Marie-Luise; Hos, Deniz; Masli, Sharmila; Bock, Felix; Cursiefen, Claus

2015-06-01

In the murine cornea, which is an established model for analyzing pathologic lymphatic vessel growth, phenotypic heterogeneity of the endogenous lymphatic vessels in the limbus of the cornea was previously described. In this study, the cornea of BALB/c, C57BL/6, and FVB mice with different limbal lymphangiogenic phenotypes was analyzed to identify novel candidates potentially influencing lymphatic vessel growth. Pathway specific expression analysis of the cornea was performed to identify novel candidate genes. Corneal protein expression of the respective candidates was analyzed by fluorescent immunohistochemistry. The effect of the candidates on proliferation of human dermal lymphatic endothelial cells (HDLECs) was analyzed by BrdU proliferation ELISA. Thirteen genes were differentially regulated in corneas of mouse strains with more endogenous limbal lymphatic vessels (high-lymphangiogenic) (C57BL/6) compared to mouse strains with less endogenous limbal lymphatic vessels (low-lymphangiogenic) (BALB/c, FVB). Two candidates, Tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/Trail) and Plasminogen activator, tissue (Plat/tPA) were expressed in the cornea of BALB/c and C57BL/6 mice on the protein level. In vitro, Trail and recombinant tPA inhibited the proliferation of human dermal lymphatic endothelial cells. Molecular analysis of the naive cornea in mouse strains with different limbal lymphatic phenotypes is a valuable model to identify novel endogenous regulators of lymphangiogenesis.

Diagnostic investigation of porcine periweaning failure-to-thrive syndrome: lack of compelling evidence linking to common porcine pathogens.

Science.gov (United States)

Huang, Yanyun; Gauvreau, Henry; Harding, John

2012-01-01

Porcine periweaning failure-to-thrive syndrome (PFTS), an increasingly recognized syndrome in the swine industry of North America, is characterized by the anorexia of nursery pigs noticeable within 1 week of weaning, and progressive loss of body condition and lethargy during the next 1-2 weeks. Morbidity caused by PFTS is moderate, but case fatality is high. The etiology of PFTS is presently unknown and may include infectious agent(s), noninfectious factors, or both. PFTS was identified in a high health status farm with good management in early 2007. A diagnostic investigation was undertaken to identify the pathological lesions of, and infectious agents associated with, pigs demonstrating typical clinical signs. Affected (PFTS-SICK) and unaffected (PFTS-HLTHY) pigs from an affected farm, and unaffected pigs from 2 unaffected farms, were examined. The most prevalent lesions in PFTS-SICK pigs were superficial lymphocytic fundic gastritis, atrophic enteritis, superficial colitis, lymphocytic and neutrophilic rhinitis, mild nonsuppurative meningoencephalitis, and thymic atrophy. Rotavirus A and Betacoronavirus 1 (Porcine hemagglutinating encephalomyelitis virus) were identified only in PFTS-SICK pigs, but the significance of the viruses is uncertain because PFTS is not consistent with the typical presentation following infection by these pathogens. Porcine reproductive and respiratory syndrome virus, Porcine circovirus-2, Influenza A virus, Alphacoronavirus 1 (Transmissible gastroenteritis virus), Torque teno virus 1, Brachyspira hyodysenteriae, and Brachyspira pilosicoli were not identified in PFTS-SICK pigs. Suid herpesvirus 2 (Porcine cytomegalovirus), Porcine enteric calicivirus, Torque teno virus 2, pathogenic Escherichia coli, and coccidia were detected in both PFTS-SICK and PFTS-HLTHY pigs. It was concluded that there is a lack of compelling evidence that PFTS is caused by any of these pathogens.

Experience of Comamonas acidovorans keratitis with delayed onset and treatment response in immunocompromised cornea.

Science.gov (United States)

Lee, Sang Mok; Kim, Mee Kum; Lee, Jae Lim; Wee, Won Ryang; Lee, Jin Hak

2008-03-01

To report 2 cases of Comamonas acidovorans keratitis in immunocompromised cornea. A complete review of the medical records of the two cases of Comamonas acidovorans keratitis. We found some similarities in clinical courses of two cases. Both of them showed development of keratitis during the management with corticosteroids, delayed onset, slow response to antibiotics, and relatively less affected corneal epithelium. Comamonas acidovorans is known as a less virulent organism. However it can cause an indolent infection that responds slowly even to adequate antibiotics therapy in immunocompromised corneas.

Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

Science.gov (United States)

Silverman, Erika; Zhao, Jin; Merriam, John C; Nagasaki, Takayuki

2017-02-01

Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

A comparison study of Riboflavin/UV-A and Rose-Bengal/Green light cross-linking of the rabbit corneas using optical coherence elastography

Science.gov (United States)

Li, Jiasong; Singh, Manmohan; Han, Zhaolong; Vantipalli, Srilatha; Liu, Chih-Hao; Wu, Chen; Raghunathan, Raksha; Kazemi, Tina; Twa, Michael D.; Larin, Kirill V.

2016-03-01

The biomechanical properties of the cornea are critical factors which determine its health and subsequent visual acuity. Keratoconus is a structural degeneration of the cornea which can diminish vision quality. Riboflavin/UV-A corneal collagen cross-linking (UV-CXL) is an emerging treatment that increases the stiffness of the cornea and improves its ability to resist further degeneration. While UV-CXL has shown great promise for effective therapy of the keratoconus, there are concerns associated with the UV irradiation, such as keratocyte cytotoxicity. Rose-bengal/green light corneal collagen cross-linking (RGX) has been proposed as an alternative to UV-CXL. Because of the high absorbance of the rose-bengal dye at green wavelengths, the treatment time is significantly shorter than with UV-CXL. Moreover, because green light is used in lieu of UV irradiation, there are no cytotoxic side-effects. In this study, noncontact optical coherence elastography (OCE) was used to compare the outcomes of UV-CXL and RGX treatment in rabbit cornea. Low-amplitude (micrometer scale) elastic waves were induced by a focused air-pulse loading system. The elastic wave propagation was then imaged by a phase-stabilized swept source OCE (PhS-SSOCE) system. The changes in the viscoelasticity of the corneas were quantified by a previously developed modified Rayleigh Lamb frequency model. The depth-resolved micro-scale phase-velocity distribution in the cornea was used to reveal the depth-wise heterogeneity before and after both cross-linking techniques. Our results show that UV-CXL and RGX increased the stiffness of the corneas by ~54% and ~5% while reducing the viscosity by ~42% and ~17%, respectively. The depth-wise phase velocities showed that UV-CXL affected the anterior ~1/3 of the corneas, while RGX only affected the anterior ~1/7 of the corneas.

Effects of acetylcholine (ACh) and norepinephrine (NE) on phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in rabbit cornea

International Nuclear Information System (INIS)

Akhtar, R.A.; Abdel-Latif, A.A.

1986-01-01

Muscarinic cholinergic and α 1 -adrenergic agonists provoke hydrolysis of PIP 2 into diacylglycerol (DG) and inositol trisphosphate (IP 3 ) in a wide variety of tissue. Recently, IP 3 has been shown to mobilize Ca 2+ from ER in several permeabilized tissue preparations. Although rabbit cornea is enriched in ACh and NE, the physiological function of these neurotransmitters is unclear. The present studies were initiated to determine the effects of cholinergic and adrenergic agonists on PIP 2 turnover in the cornea. Addition of ACh or NE (50 μM each) to the 32 P-labeled corneas for 10 min decreased the radioactivity in PIP 2 by 33 and 36%, and increased the radioactivity in phosphatidic acid by 72 and 52%, respectively. When the corneas were labeled with myo-[ 3 H]inositol, ACh and NE increased the accumulation of IP 3 by 92 and 48%, respectively. The effects of ACh and NE on phospholipid labeling and IP 3 accumulation were specifically inhibited by atropine (10 μM) and prazosin (10 μM), respectively. The data suggest the presence of muscarinic cholinergic and α 1 -adrenergic receptors in the rabbit cornea. Furthermore, activation of these receptors leads to cleavage of PIP 2 into DG and IP 3 which may function as second messengers in this tissue

Taurine effect on cytogenetic lesions in the cornea of mice exposed to 9 Gev proton irradiation

International Nuclear Information System (INIS)

Vorozhtsova, S.V.; Yartsev, E.I.

1989-01-01

Possibilities of preventive measures and treatment of cytogenetic injuries in the mice cornea, subjected to proton irradiation at 9 Gev were studied. Taurine containing solution (TCS) was used as a radiomodifying agent. It is shown that TCS application enables to decrease aberrant mitoses level in cornea epithelium cells of mice. Antiactinic effect of the above agent is determined by its considerable action on mitotic delay

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

Directory of Open Access Journals (Sweden)

Christopher A Smith

Full Text Available To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC. BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1 concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Second harmonic generation microscopy of the living human cornea

Science.gov (United States)

Artal, Pablo; Ávila, Francisco; Bueno, Juan

2018-02-01

Second Harmonic Generation (SHG) microscopy provides high-resolution structural imaging of the corneal stroma without the need of labelling techniques. This powerful tool has never been applied to living human eyes so far. Here, we present a new compact SHG microscope specifically developed to image the structural organization of the corneal lamellae in living healthy human volunteers. The research prototype incorporates a long-working distance dry objective that allows non-contact three-dimensional SHG imaging of the cornea. Safety assessment and effectiveness of the system were firstly tested in ex-vivo fresh eyes. The maximum average power of the used illumination laser was 20 mW, more than 10 times below the maximum permissible exposure (according to ANSI Z136.1-2000). The instrument was successfully employed to obtain non-contact and non-invasive SHG of the living human eye within well-established light safety limits. This represents the first recording of in vivo SHG images of the human cornea using a compact multiphoton microscope. This might become an important tool in Ophthalmology for early diagnosis and tracking ocular pathologies.

Tachykinins in the porcine pancreas

DEFF Research Database (Denmark)

Schmidt, P T; Tornøe, K; Poulsen, Steen Seier

2000-01-01

The localization, release, and effects of substance P and neurokinin A were studied in the porcine pancreas and the localization of substance P immunoreactive nerve fibers was examined by immunohistochemistry. The effects of electrical vagus stimulation and capsaicin infusion on tachykinin release...... and the effects of substance P and neurokinin A infusion on insulin, glucagon, somatostatin, and exocrine secretion were studied using the isolated perfused porcine pancreas with intact vagal innervation. NK-1 and NK-2 receptor antagonists were used to investigate receptor involvement. Substance P immunoreactive...

Methods for the detection and serum depletion of porcine galectin-3.

Science.gov (United States)

Eliaz, Isaac; Patil, Aarti; Navarro-Alvarez, Nalu; Wang, Zhirui; Eliaz, Amity; Weil, Elaine; Wilk, Barry; Sachs, David H; Huang, Christene A

2017-10-01

Circulating galectin-3 (Gal-3) is elevated in systemic inflammatory disorders, fibrotic diseases, and in cancers. Gal-3 is a promising cancer target where it promotes tumorigenesis and metastasis, as well as in renal, pulmonary, hepatic, and cardiovascular diseases, because of its role as a driver of fibrotic remodeling. This reports goal was to establish methods for the detection and removal of porcine Gal-3 that will enable further studies of the therapeutic potential of Gal-3 depletion by apheresis in porcine disease models. The long-term aim is to develop a safe, effective method of removing Gal-3 via apheresis as a standalone therapeutic tool and as an adjuvant to other therapies. Purified recombinant porcine Gal-3 was prepared and used as the standard for development of a porcine Gal-3 enzyme-linked immunosorbent assay (ELISA). Different affinity column matrices that incorporated either a rat IgG2a anti-Gal-3 monoclonal antibody or carbohydrate ligand were assessed for depletion of Gal-3 from porcine serum. A porcine Gal-3 ELISA with a linear range from 0.3 to 20 ng/mL was able to detect native porcine Gal-3 in both fetal (∼150-200 ng/mL) and juvenile (∼5-15 ng/mL) porcine serum samples. Use of an anti-Gal-3 monoclonal antibody affinity column depleted Gal-3 from porcine serum to at least 313 pg/mL, the limit of ELISA detection. Methods have been developed for the detection and depletion of porcine Gal-3. These methods will be used to study the specific effects of Gal-3 depletion via apheresis in porcine models of disease. © 2017 Wiley Periodicals, Inc.

Lentiviral Vector Gene Transfer to Porcine Airways

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Patrick L Sinn

2012-01-01

Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

Expression profiles of aquaporins in rat conjunctiva, cornea, lacrimal gland and Meibomian gland.

Science.gov (United States)

Yu, Dongfang; Thelin, William R; Randell, Scott H; Boucher, Richard C

2012-10-01

The aim of the study was to elucidate aquaporin (AQP) family member mRNA expression and protein expression/localization in the rat lacrimal functional unit. The mRNA expression of all rat AQPs (AQP0-9, 11-12) in palpebral, fornical, and bulbar conjunctiva, cornea, lacrimal gland, and Meibomian gland was measured by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and real time RT-PCR. Antibodies against AQP1, 3, 4, 5, 9, and 11 were used in Western blotting and immunohistochemistry to determine protein expression and distribution. Our study demonstrated characteristic AQP expression profiles in rat ocular tissues. AQP1, 3, 4, 5, 8, 9, 11, and 12 mRNA were detected in conjunctiva. AQP0, 1, 2, 3, 4, 5, 6, 11, and 12 mRNA were expressed in cornea. AQP0, 1, 2, 3, 4, 5, 7, 8, and 11 mRNA were detected in lacrimal gland. AQP1, 3, 4, 5, 7, 8, 9, 11, and 12 mRNA were identified in Meibomian gland. By Western blot, AQP1, 3, 5, and 11 were detected in conjunctiva; AQP1, 3, 5, and 11 were identified in cornea; AQP1, 3, 4, 5, and 11 were detected in lacrimal gland; and AQP1, 3, 4, 5, 9, and 11 were present in Meibomian gland. Immunohistochemistry localized AQPs to distinct sites in the various tissues. This study rigorously analyzed AQPs expression and localization in rat conjunctiva, cornea, lacrimal gland, and Meibomian gland tissues. Our findings provide a comprehensive platform for further investigation into the physiological or pathophysiological relevance of AQPs in ocular surface. Copyright © 2012 Elsevier Ltd. All rights reserved.

Study of the protective properties of paraaminobenzoic acid for cornea of adult rats under X-radiation

International Nuclear Information System (INIS)

Stroeva, O.G.; Panova, I.G.; Mel'nikova, I.I.

1997-01-01

To test the efficiency of para-aminobenzoic acid (PABA) as a radioprotector for mammal tissues the protective properties of PABA for cornea of adult rats-males exposed to single whole-body irradiation were studied. X-irradiation was performed using RUM-17 facility at the dose of 5 Gy (dose rate is of 0.886 Gy/min). Results obtained prove reliably radioprotective and therapeutic effect of PABA on the cornea cells [ru

Progress, problems and prospects of porcine pluripotent stem cells

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Hanning WANG,Yangli PEI,Ning LI,Jianyong HAN

2014-02-01

Full Text Available Pluripotent stem cells (PSCs, including embryonic stem cells (ESCs and induced PSCs (iPSCs, can differentiate into cells of the three germ layers, suggesting that PSCs have great potential for basic developmental biology research and wide applications for clinical medicine. Genuine ESCs and iPSCs have been derived from mice and rats, but not from livestock such as the pig─an ideal animal model for studying human disease and regenerative medicine due to similarities with human physiologic processes. Efforts to derive porcine ESCs and iPSCs have not yielded high-quality PSCs that can produce chimeras with germline transmission. Thus, exploration of the unique porcine gene regulation network of preimplantation embryonic development may permit optimization of in vitro culture systems for raising porcine PSCs. Here we summarize the recent progress in porcine PSC generation as well as the problems encountered during this progress and we depict prospects for generating porcine naive PSCs.

The filtering, clear-cornea diathermal keratostomy: a minor Danish multicenter study

DEFF Research Database (Denmark)

Kessing, S.V.; Nissen, O.I.; Thygesen, J.

2008-01-01

PURPOSE: Is the new micropenetrating, clear-cornea procedure, intrastromal diathermal keratostomy (IDK), an alternative to the intricate "modern trabeculectomy"? METHODS: Prospective multicenter study. Four surgeons from 4 Danish eye departments attended an IDK course and subsequently decided whe...

Porcine circovirus diseases

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Ristoski Trpe

2009-05-01

Full Text Available Porcine circovirus type 2 belongs on the family Circoviridae. This virus family includes small, non-enveloped viruses, with a circular, single-standed DNA genome.This virus causes mainly subclinical infections, but a number of diseases have been linked to it (porcine circovirus diseases, PCVD. The most economically important PCVD is postweaning multisystemic wasting syndrome (PMWS, which mainly affects pigs of 2 to 5 months of age, with progressive wasting, diarrhea and respiratory disorders. Main PMWS lesions are found in lymphoid tissues, which are characterized by lymphocyte depletion with granulomatous (histiocytic and multinucleate giant cell infiltration. PMWS is considered as multifactorial disease, with a number of infectious and non-infectious factors able to act as disease triggering in PCV2 infected pigs. PCVDs are worldwide distributed, and PMWS was diagnosed in Macedonia in 2007.

Deciphering the porcine intestinal microRNA transcriptome

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Keller Andreas

2010-04-01

Full Text Available Abstract Background While more than 700 microRNAs (miRNAs are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. Results Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. Conclusions In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.

Damage Threshold of In Vivo Rabbit Cornea by 2 micron Laser Irradiation

National Research Council Canada - National Science Library

Chen, Bo; Oliver, Jeffery; Dutta, Soumak; Rylander, III, Grady H; Thomsen, Sharon L; Welch, Ashley J

2007-01-01

To support refinement of the Maximum Permissible Exposure (MPE) safety limits, a series of experiments were conducted in vivo on Dutch Belted rabbit corneas to determine corneal minimum visible lesion thresholds...

Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes.

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Lan, H N; Hong, P; Li, R N; Shan, A S; Zheng, X

2017-10-01

The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal. Copyright © 2017 Elsevier Inc. All rights reserved.

Dexamethasone Drug Eluting Nanowafers Control Inflammation in Alkali-Burned Corneas Associated With Dry Eye

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Bian, Fang; Shin, Crystal S.; Wang, Changjun; Pflugfelder, Stephen C.; Acharya, Ghanashyam; De Paiva, Cintia S.

2016-01-01

Purpose To evaluate the efficacy of a controlled release dexamethasone delivery system for suppressing inflammation in an ocular burn + desiccating stress (OB+DS) model. Methods Nanowafers (NW) loaded with Dexamethasone (Dex, 10 μg) or vehicles (2.5% Methylcellulose; MC) were fabricated using hydrogel template strategy. C57BL/6 mice were subjected to unilateral alkali ocular burn with concomitant desiccating stress for 2 or 5 days and topically treated either with 2 μL of 0.1% Dex or vehicle four times per day and compared with mice that had MC-NW or Dex-NW placed on their corneas. Clinical parameters were evaluated daily. Mice were euthanized after 2 or 5 days. Quantitative PCR evaluated the expression of inflammatory cytokines IL-1β and IL-6 and matrix metalloproteinases (MMP) in whole cornea lysates. Myeloperoxidase activity (MPO) was measured using a commercial kit in cornea lysates. Results Both Dex drop and Dex-NW groups had significantly lower corneal opacity scores compared with their vehicles. Both Dex drops and Dex-NW significantly decreased expression of IL-1β, IL-6, and MMP-9 RNA transcripts compared with vehicle drops or wafers 2 and 5 days after the initial lesion. A significant lower number of neutrophils was found in both Dex treatment groups and this was accompanied by decreased MPO activity compared with vehicle controls. Conclusions Dex-NW has efficacy equal to Dex drops in preserving corneal clarity and decreasing expression of MMPs and inflammatory cytokines of the corneas of mice subjected to an OB+DS model. PMID:27327581

Femtosecond laser cutting of human corneas for the subbasal nerve plexus evaluation.

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Kowtharapu, B S; Marfurt, C; Hovakimyan, M; Will, F; Richter, H; Wree, A; Stachs, O; Guthoff, R F

2017-01-01

Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon-a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70-90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti-neuronal β-III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20-30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Sequence conservation between porcine and human LRRK2

DEFF Research Database (Denmark)

Larsen, Knud; Madsen, Lone Bruhn

2009-01-01

 Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved...... and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson's disease...

Specific demographic factors could predict deceased potential cornea donors: A retrospective study from Beijing Tongren Hospital Eye Bank.

Science.gov (United States)

Liu, Yang; Zhang, Yingnan; Zhang, Jing; Yu, Jin; Liang, Qingfeng; Pan, Zhiqiang

2017-12-01

Compared with evident cornea donors (ECDs), deceased potential cornea donors (DPCDs) have no obvious donor identifications to reference, which causes many eligible cornea tissues to be wasted. The demographic characteristics of DPCDs might be different from those of ECDs owing to the following different features: donation consent provided by relatives and willingness to donate before death. Thus, the aim of this study is to reveal the demographic characteristics of DPCDs by comparing DPCDs and ECDs.The demographic factors of 138 donors (both DPCDs and ECDs) were collected from the Beijing Tongren Hospital Eye Bank database and analyzed. To differentiate DPCDs from ECDs using the above-mentioned features, we interviewed the relatives of the donors by telephone. The relatives' attitudes toward cornea donation and their suggestions for our donation service were also acquired during the interview. Two logistic regressions were performed to reveal the demographic factors influencing the 2 features and indicate DPCDs.The donors had certain demographic characteristics (elderly, secondary, or tertiary education level, central district resident), and the most frequent cause of death for the donors was a malignant tumor (n = 56, 43.1%). All the relatives had positive attitudes toward cornea donations, and they hoped to increase publicity efforts to encourage more people to donate and establish more convenient and efficient access for cornea donation. In univariate regressions, age (P = .004, >50 years: odds ratio [OR] = 6.89, 95% confidence interval [CI]: 1.82-26.05), marital status (P = .043, divorced: OR = 9.00,95% CI: 1.33-60.80) significantly influenced relative consent, whereas age (P = .001, >50 years: OR = 15.00, 95% CI: 3.00-74.98), and family address (P = .001, central district: OR = 1) were significant factors influencing the willingness to donate before death. In multivariate regression, age (P = .021, >50 years: OR = 8

3D Characterization of corneal deformation using ultrasound speckle tracking

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Keyton Clayson

2017-11-01

Full Text Available The three-dimensional (3D mechanical response of the cornea to intraocular pressure (IOP elevation has not been previously reported. In this study, we use an ultrasound speckle tracking technique to measure the 3D displacements and strains within the central 5.5mm of porcine corneas during the whole globe inflation. Inflation tests were performed on dextran-treated corneas (treated with a 10% dextran solution and untreated corneas. The dextran-treated corneas showed an inflation response expected of a thin spherical shell, with through-thickness thinning and in-plane stretch, although the strain magnitudes exhibited a heterogeneous spatial distribution from the central to more peripheral cornea. The untreated eyes demonstrated a response consistent with swelling during experimentation, with through-thickness expansion overriding the inflation response. The average volume ratios obtained in both groups was near 1 confirming general incompressibility, but local regions of volume loss or expansion were observed. These results suggest that biomechanical measurements in 3D provide important new insight to understand the mechanical response of ocular tissues such as the cornea.

First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus.

Science.gov (United States)

Schirtzinger, Erin E; Suddith, Andrew W; Hause, Benjamin M; Hesse, Richard A

2015-10-16

Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 (porcine bocavirus 3, 4-1 and 4-2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure. Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples. Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6-99.5 % identity with the North American strains) to be the North American strains nearest relative. These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is

Infrared irradiation alters the expression of matrix metalloproteinases and glycosaminoglycans in the cornea and crystalline lens.

Science.gov (United States)

Dadoukis, Panagiotis; Klagas, Ioannis; Komnenou, Anastasia; Karakiulakis, George; Karoutis, Athanasios; Karampatakis, Vassilios; Papakonstantinou, Eleni

2013-08-01

Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens. New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1% diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes. Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation. The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.

[Biological availability of ophthalmic drugs. 1. Increasing drug permeability in the cornea].

Science.gov (United States)

Masteiková, R; Chalupová, Z; Savickiene, N

2004-03-01

Most eye diseases are treated by topical administration of ophthalmic preparations. Low ophthalmic bioavailability is due to a number of physiological and physicochemical factors. The main obstacle to the penetration of active ingredients to the eye is the layered character of the cornea. Formulation of ophthalmic preparations enabling easier penetration of this biological barrier thus ranks among the most effective ways of improving bioavailability. Penetrability of the cornea can be increased by the following methods: a) adjustment of the actual acidity in such a way that pH of the preparation makes it possible to produce the optimal portion of non-ionized particles; b) incorporation of absorption enhancers (non-ionic tensides, salts of bile acids, some antimicrobial substances, EDTA, cyclodextrins, etc.) into the composition of the preparation, and c) production of prodrugs or ionic pairs.

Corneal Collagen Cross-Linking with Hypoosmolar Riboflavin Solution in Keratoconic Corneas

Directory of Open Access Journals (Sweden)

Shaofeng Gu

2014-01-01

Full Text Available Purpose. To report the 12-month outcomes of corneal collagen cross-linking (CXL with a hypoosmolar riboflavin and ultraviolet-A (UVA irradiation in thin corneas. Methods. Eight eyes underwent CXL using a hypoosmolar riboflavin solution after epithelial removal. The corrected distance visual acuity (CDVA, manifest refraction, the mean thinnest corneal thickness (MTCT, and the endothelial cell density (ECD were evaluated before and 6 and 12 months after CXL. Results. The MTCT was 413.9 ± 12.4 μm before treatment and reduced to 381.1 ± 7.3 μm after the removal of the epithelium. After CXL, the thickness decreased to 410.3 ± 14.5 μm at the last follow-up. Before treatment, the mean K-value of the apex of the keratoconus corneas was 58.7 ± 3.5 diopters and slightly decreased (57.7 ± 4.9 diopters at 12 months. The mean CDVA was 0.54 ± 0.23 logarithm of the minimal angle of resolution before treatment and increased to 0.51 ± 0.21 logarithm at the last follow-up. The ECD was 2731.4 ± 191.8 cells/mm2 before treatment and was 2733.4 ± 222.6 cells/mm2 at 12 months after treatment. Conclusions. CXL with a hypoosmolar riboflavin in thin corneas seems to be a promising method for keratoconic eyes with the mean thinnest corneal thickness less than 400 μm without epithelium.

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Science.gov (United States)

Di Tommaso, Claudia; Bourges, Jean-Louis; Valamanesh, Fatemeh; Trubitsyn, Gregory; Torriglia, Alicia; Jeanny, Jean-Claude; Behar-Cohen, Francine; Gurny, Robert; Möller, Michael

2012-06-01

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

OpenAIRE

Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2016-01-01

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. I...

In vivo imaging through the entire thickness of human cornea by full-field optical coherence tomography

Science.gov (United States)

Mazlin, Viacheslav; Xiao, Peng; Dalimier, Eugénie; Grieve, Kate; Irsch, Kristina; Sahel, José; Fink, Mathias; Boccara, Claude

2018-02-01

Despite obvious improvements in visualization of the in vivo cornea through the faster imaging speeds and higher axial resolutions, cellular imaging stays unresolvable task for OCT, as en face viewing with a high lateral resolution is required. The latter is possible with FFOCT, a method that relies on a camera, moderate numerical aperture (NA) objectives and an incoherent light source to provide en face images with a micrometer-level resolution. Recently, we for the first time demonstrated the ability of FFOCT to capture images from the in vivo human cornea1. In the current paper we present an extensive study of appearance of healthy in vivo human corneas under FFOCT examination. En face corneal images with a micrometer-level resolution were obtained from the three healthy subjects. For each subject it was possible to acquire images through the entire corneal depth and visualize the epithelium structures, Bowman's layer, sub-basal nerve plexus (SNP) fibers, anterior, middle and posterior stroma, endothelial cells with nuclei. Dimensions and densities of the structures visible with FFOCT, are in agreement with those seen by other cornea imaging methods. Cellular-level details in the images obtained together with the relatively large field-of-view (FOV) and contactless way of imaging make this device a promising candidate for becoming a new tool in ophthalmological diagnostics.

Spatial clustering of porcine cysticercosis in Mbulu district, northern Tanzania.

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Helena A Ngowi

Full Text Available BACKGROUND: Porcine cysticercosis is caused by a zoonotic tapeworm, Taenia solium, which causes serious disease syndromes in human. Effective control of the parasite requires knowledge on the burden and pattern of the infections in order to properly direct limited resources. The objective of this study was to establish the spatial distribution of porcine cysticercosis in Mbulu district, northern Tanzania, to guide control strategies. METHODOLOGY/PRINCIPAL FINDINGS: This study is a secondary analysis of data collected during the baseline and follow-up periods of a randomized community trial aiming at reducing the incidence rate of porcine cysticercosis through an educational program. At baseline, 784 randomly selected pig-keeping households located in 42 villages in 14 wards were included. Lingual examination of indigenous pigs aged 2-12 (median 8 months, one randomly selected from each household, were conducted. Data from the control group of the randomized trial that included 21 of the 42 villages were used for the incidence study. A total of 295 pig-keeping households were provided with sentinel pigs (one each and reassessed for cysticercosis incidence once or twice for 2-9 (median 4 months using lingual examination and antigen ELISA. Prevalence of porcine cysticercosis was computed in Epi Info 3.5. The prevalence and incidence of porcine cysticercosis were mapped at household level using ArcView 3.2. K functions were computed in R software to assess general clustering of porcine cysticercosis. Spatial scan statistics were computed in SatScan to identify local clusters of the infection. The overall prevalence of porcine cysticercosis was 7.3% (95% CI: 5.6, 9.4; n = 784. The K functions revealed a significant overall clustering of porcine cysticercosis incidence for all distances between 600 m and 5 km from a randomly chosen case household based on Ag-ELISA. Lingual examination revealed clustering from 650 m to 6 km and between 7.5 and 10 km

Porcine SLITRK1

DEFF Research Database (Denmark)

Larsen, Knud Erik; Momeni, Jamal; Farajzadeh, Leila

2014-01-01

The membrane protein SLITRK1 functions as a developmentally regulated stimulator of neurite outgrowth and variants in this gene have been implicated in Tourette syndrome. In the current study we have cloned and characterized the porcine SLITRK1 gene. The genomic organization of SLITRK1 lacks...

A human hemi-cornea model for eye irritation testing: quality control of production, reliability and predictive capacity.

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Engelke, M; Zorn-Kruppa, M; Gabel, D; Reisinger, K; Rusche, B; Mewes, K R

2013-02-01

We have developed a 3-dimensional human hemi-cornea which comprises an immortalized epithelial cell line and keratocytes embedded in a collagen stroma. In the present study, we have used MTT reduction of the whole tissue to clarify whether the production of this complex 3-D-model is transferable into other laboratories and whether these tissues can be constructed reproducibly. Our results demonstrate the reproducible production of the hemi-cornea model according to standard operation procedures using 15 independent batches of reconstructed hemi-cornea models in two independent laboratories each. Furthermore, the hemi-cornea tissues have been treated with 20 chemicals of different eye-irritating potential under blind conditions to assess the performance and limitations of our test system comparing three different prediction models. The most suitable prediction model revealed an overall in vitro-in vivo concordance of 80% and 70% in the participating laboratories, respectively, and an inter-laboratory concordance of 80%. Sensitivity of the test was 77% and specificity was between 57% and 86% to discriminate classified from non-classified chemicals. We conclude that additional physiologically relevant endpoints in both epithelium and stroma have to be developed for the reliable prediction of all GHS classes of eye irritation in one stand alone test system. Copyright © 2012 Elsevier Ltd. All rights reserved.

The healing of alkali-injured cornea is stimulated by a novel matrix regenerating agent (RGTA, CACICOL20): a biopolymer mimicking heparan sulfates reducing proteolytic, oxidative and nitrosative damage.

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Cejkova, Jitka; Olmiere, Celine; Cejka, Cestmir; Trosan, Peter; Holan, Vladimir

2014-04-01

The efficacy of a chemically modified dextran - heparan sulfate mimicking regenerating agent (RGTA) on the healing of the rabbit cornea injured with alkali was examined. The eyes were injured with 0.15 N NaOH applied on the cornea or with 1.0 N NaOH using a 8 mm diameter filter paper disk. Then RGTA or placebo was applied on the cornea. In the last group of rabbits, corneas injured with the high alkali concentration were left without any treatment for four weeks; subsequently, the corneas were treated with RGTA or placebo. The central corneal thickness was measured using a pachymeter. The corneas were examined morphologically, immunohistochemically and for real time-PCR. Compared to control (unaffected) corneas, following the application of low alkali concentration the expression of urokinase-type plasminogen activator, metalloproteinase 9, nitric oxide synthase and xanthine oxidase was increased in the injured corneal epithelium of placebo-treated eyes, whereas the expression of antioxidant enzymes was reduced. Nitrotyrosine and malondialdehyde stainings appeared in the corneal epithelium. RGTA application suppressed the antioxidant/prooxidant imbalance and reduced the expression of the above-mentioned immunohistochemical markers. The corneal thickness increased after alkali injury, decreased during corneal healing after RGTA treatment faster than after placebo application. Following the injury with the high alkali concentration, corneal inflammation and neovascularization were highly pronounced in placebo-treated corneas, whereas in RGTA-treated corneas they were significantly supressed. When RGTA or placebo application was started later after alkali injury and corneas were ulcerated, subsequent RGTA treatment healed the majority of them. In conclusion, RGTA facilitates the healing of injured corneas via a reduction of proteolytic, oxidative and nitrosative damage.

Donor cornea procurement: six-year review of the role of the eye bank in South Australia.

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Williams, K A; White, M A; Badenoch, P R; Wedding, T R; Alfrich, S J; Sawyer, M A; Noack, L M; Johnstone, E W; Zilm, G; Coster, D J

1990-02-01

The Lions Eye Bank of South Australia was established six years ago and has collected corneas from 790 donors. The consent rate is currently 82% of requests made. Two-thirds of donors have been male, with mean donor age/year varying from 54 to 64 years (range two to 93 years). Cardiovascular and respiratory diseases, trauma and haemorrhage account for 80% of all donor deaths. Mean death to enucleation time is five hours. Corneas assessed as being of excellent or very good quality are released preferentially from the bank; those with central endothelial cell counts of less than 1500 cells/mm2 are discarded. Fewer than 1% of donors have returned a positive result for HIV or hepatitis B. Of the 1580 corneas collected by the bank, 863 (55%) have been used for transplantation with a primary non-function rate of 0.46%. The evolving policies, logistics of operation and methodologies employed by the bank are described in detail.

Functional verification of a porcine myostatin propeptide mutant.

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Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

Estudo comparativo entre duas plataformas para realização de Lasik personalizado para correção de miopia e astigmatismo: Alcon CustomCornea® versus Bausch & Lomb Zyoptix® Wavefront-Guided Lasik for low to moderate myopia: CustomCornea® versus Zyoptix®

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Telma Pereira Barreiro

2009-08-01

Full Text Available OBJETIVO: Comparar os resultados obtidos após o Lasik personalizado utilizando duas plataformas diferentes. MÉTODOS: Estudo prospectivo, randomizado com 50 pacientes míopes submetidos a cirurgia refrativa em ambos os olhos. Foram selecionados para o estudo, pacientes com equivalente esférico semelhante entre os olhos. Todos foram submetidos a Lasik bilateral e simultâneo, sendo que um olho foi operado pela plataforma CustomCornea® e o outro pela Zyoptix®. Acuidade visual sem e com correção, refração dinâmica e estática, medida das aberrações oculares, teste de sensibilidade ao contraste foram realizados no período pré-operatório e pós-operatório de 1, 3 e 6 meses. RESULTADOS: No período pré-operatório a média do equivalente esférico era de -3,29 ± 1,56 D no grupo CustomCornea® e de -3,22 ± 1,50 D no Zyoptix® (p=0,267. No sexto mês de pós-operatório, a média do equivalente esférico no grupo CustomCornea® era de -0,077 ± 0,23 D e -0,282 ± 0,30 D no Zyoptix® (p 20/20 foi alcançada em 86% dos olhos no grupo CustomCornea® e 70% no grupo Zyoptix® (p=0,094. Nenhum paciente perdeu duas ou mais linhas da melhor acuidade visual corrigida. Cem por cento dos olhos CustomCornea® e 88% dos Zyoptix® ficaram entre ± 0,50 D da emetropia (p=0,014*. Melhora da sensibilidade ao contraste em todas as frequências espaciais testadas foi observada em ambos os grupos. A aberração esférica apresentou aumento em ambos os grupos, porém este foi estatisticamente maior na plataforma Zyoptix® (pPURPOSE: To compare the visual and clinical outcomes of Wavefront-guided laser in situ keratomileusis (Lasik with Alcon CustomCornea® and Zyoptix® systems. METHODS: A prospective, randomized, masked and bilateral study was conducted. Fifty patients with preoperative spherical equivalent ranging from -1.00 to -6.50 D were enrolled for customized ablation in both eyes. All of them were submitted to Lasik CustomCornea® treatment in one

Adjunctive cryotherapy for pigmentary keratitis in dogs: a study of 16 corneas.

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Azoulay, Thierry

2014-07-01

To assess whether soft cryotherapy (dimethylether, isobutene, and propane) can remove pigmentation of the cornea that has accumulated under different conditions when conventional therapy has been unsuccessful. Nine dogs with unilateral or bilateral corneal pigmentation (16 eyes) were included based on progressive corneal pigmentation that was unresponsive to long-term etiological treatment. The dogs had keratoconjunctivitis sicca or chronic superficial keratitis. A cryogen of 95% dimethylether, 3% isobutane, and 2% propane was applied to the pigmented areas of each cornea under anesthesia. Initial corneal pigmentation and changes were documented over the entire study period using a grading scheme and clinical photographs. Most of the pigment deposits were gone by 5-15 days after cryosurgery. Postoperatively, the dogs showed some corneal edema and corneo-conjunctival inflammation, and three dogs had superficial corneal ulcers; these symptoms had resolved by 1 month after the procedure. Follow-up for more than 90 days was available in five dogs (nine corneas), and we observed total or partial repigmentation when the underlying disease was not controlled. A new cryotherapy procedure was successfully performed in two of these dogs. Given the sensitivity to cold of melanocytes, cryotherapy is a viable adjunctive treatment for refractory severe corneal pigmentation. Etiological treatment remains necessary to prevent pigmentation from rapidly reappearing. Only a few dogs were followed for more than 90 days; further study is necessary to evaluate the long-term safety and efficacy of soft cryotherapy. © 2013 American College of Veterinary Ophthalmologists.

Targeted Porcine Genome Engineering with TALENs

DEFF Research Database (Denmark)

Luo, Yonglun; Lin, Lin; Golas, Mariola Monika

2015-01-01

confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs......, including construction of sequence-specific TALENs, delivery of TALENs into primary porcine fibroblasts, and detection of TALEN-mediated cleavage, is described. This chapter is useful for scientists who are inexperienced with TALEN engineering of porcine cells as well as of other large animals....

Extracellular matrix hydrogels from decellularized tissues: Structure and function.

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Saldin, Lindsey T; Cramer, Madeline C; Velankar, Sachin S; White, Lisa J; Badylak, Stephen F

2017-02-01

Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed. More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Finite element analysis of blunt foreign body impact on the cornea after PRK and LASIK.

Science.gov (United States)

Mousavi, Seyed Jamaleddin; Nassiri, Nariman; Masoumi, Nafiseh; Nassiri, Nader; Majdi-N, Mercede; Farzaneh, Solmaz; Djalilian, Ali R; Peyman, Gholam A

2012-01-01

To investigate the effect of blunt foreign body impact on a human cornea after photorefractive keratectomy (PRK) and LASIK using a simulation model. Computational simulations were performed using a finite element analysis program (LS-Dyna, Livermore Software Technology Corp). The blunt foreign body was set to impact at the center of the corneal surface models (after PRK and LASIK) with thicknesses of 500, 450, 400, 350, and 300 μm. Corneal rupture was assumed to occur at a peak stress of 9.45 MPa and at a strain of 18%. The foreign body projectile was blunt in shape, made from aluminum, contained plastic-kinematic properties, and had a density of 2700 kg/m(3). The projectile was launched at the center of the cornea with velocities ranging from 20 to 60 m/s. The threshold of impact velocities creating rupture in corneal thicknesses of 500, 450, 400, 350, and 300 μm were 33, 32.8, 30.7, 27.9, and 22.8 m/s, respectively, in the PRK model. In the LASIK model, the thresholds creating rupture in the stromal bed of the corneas with thicknesses of 500, 450, 400, 350, and 300 μm were 40, 38.1, 35.6, 31.5, and 26.7 m/s, respectively. The 110-μm corneal flap in the LASIK model ruptured at all velocities. Ruptures occurred at lower velocities in the PRK cornea model than in the corneal stromal bed of the LASIK model following blunt foreign body impact. Copyright 2012, SLACK Incorporated.

Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

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Cong, Yingying; Li, Xiaoxue; Bai, Yunyun [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Lv, Xiaonan [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100090 (China); Herrler, Georg [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Enjuanes, Luis [Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid (Spain); Zhou, Xingdong [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Qu, Bo [Faculty of Life Sciences, Northeast Agricultural University, Harbin 150030 (China); Meng, Fandan [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Cong, Chengcheng [College Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161 (China); Ren, Xiaofeng; Li, Guangxing [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China)

2015-04-15

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

International Nuclear Information System (INIS)

Cong, Yingying; Li, Xiaoxue; Bai, Yunyun; Lv, Xiaonan; Herrler, Georg; Enjuanes, Luis; Zhou, Xingdong; Qu, Bo; Meng, Fandan; Cong, Chengcheng; Ren, Xiaofeng; Li, Guangxing

2015-01-01

Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs

Comparison of commercial and experimental porcine circovirus type 2 (PCV2) vaccines using a triple challenge with PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV).

Science.gov (United States)

Shen, H G; Beach, N M; Huang, Y W; Halbur, P G; Meng, X J; Opriessnig, T

2010-08-23

The efficacies of commercial porcine circovirus type 2 (PCV2) vaccines and a live PCV1-2a chimeric vaccine were compared in conventional, PCV2-positive piglets using a PCV2-porcine reproductive and respiratory syndrome virus (PRRSV)-porcine parvovirus (PPV) coinfection challenge model. Seventy-three, 2-week-old pigs were randomized into seven groups including five vaccinated and two control groups. Pigs in the vaccinated groups were vaccinated at 3 weeks (one dose) or at 3 and 6 weeks (two dose) of age. All vaccine regimens tested were effective in reducing naturally occurring PCV2 viremia at 16 weeks of age and after PCV2 challenge, demonstrating the capability of the products to induce a lasting protective immunity despite the presence of PCV2 viremia at the time of vaccination. Copyright 2010 Elsevier Ltd. All rights reserved.

Circovirose suína Porcine circovirosis: a review

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Ticiana do Nascimento França

2005-06-01

Full Text Available Por meio de revisão da literatura pertinente foram coligidos e são apresentados os principais dados relativos aos aspectos epidemiológicos, clínicos, anátomo e histopatológicos observados na infecção por Circovírus Porcino tipo 2 (PCV-2 em suínos. São abordados a Síndrome Definhante Multissistêmica dos Suínos Desmamados (SDMDS, o Tremor Congênito Suíno (TCS, a Síndrome da Nefropatia e Dermatite Porcina (SNDP, bem como outras enfermidades associadas ou correlatas, a Síndrome Respiratória e Reprodutiva Porcina (SRRP, a Pneumonia Necrotizante Proliferativa (PNP e as falhas reprodutivas. Uma vez que a SDMSD já foi registrada na Região Sul do Brasil e no Estado do Rio de Janeiro esse estudo objetiva chamar a atenção para o especial significado dessa virose para a suinocultura brasileira, em função dos prejuízos econômicos por ela determinados.The literature of Porcine Circovirosis, including the main data on epidemiology and clinical, macroscopic and microscopic alterations of the infection of swine by Porcine Circovirus type 2 (PCV-2, is reviewed. There are various forms of infection: the [Porcine] Postweaning Multisystemic Wasting Syndrome (PMWS, Porcine Congenital Tremor, Porcine Dermatitis and Nephropathy Syndrome, and other associated or correlated diseases as the Porcine Reproductive and Respiratory Syndrome, Proliferative Necrotizing Pneumonia, and reproductive disorders. As PMWS already has been reported from southern Brazil and from the state of Rio de Janeiro, the objective of this review is to draw attention to the implications of this virosis for swine production in Brazil and its economical importance.

Determination of natamycin in rabbit cornea by high-performance liquid chromatography-tandem mass spectrometry with protective soaking extraction technology.

Science.gov (United States)

Tianyang, Zhou; Ling, Zhu; Huiyun, Xia; Jijun, He; Junjie, Zhang

2014-10-15

A new selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the quantification of natamycin (NAT) in rabbit corneas with amphotericin B as the internal standard (IS). The cornea samples were processed by a simple and protective methanol soaking extraction technology. The NAT could be extracted completely from rabbit cornea after 24h of soaking with methanol under a mild condition. Chromatographic separation was performed on a C18 column (2.1mm×50mm, 3.5μm) using mobile phase with ammonium acetate buffer (pH 4.5; 4.0mM):acetonitrile (40:60, v/v) at a flow rate of 0.25ml/min. Quantification was performed using the transitions 666.2→503.2 m/z for NAT and 924.5→906.6 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring mode. The assay was validated over a concentration range of 8.64ng/ml to 843ng/ml with lower limit of detection of 4.32ng/ml. The method was validated with respect to linearity, accuracy, precision, recovery, stability and extracting efficiency. The extraction recovery of NAT from cornea samples was approximately 100% with the new methanol soaking extraction procedure. The method has been successfully applied to the ocular pharmacokinetic studies of NAT eye drops in the cornea of Japanese white rabbit. Copyright © 2014 Elsevier B.V. All rights reserved.

Measurement and modeling of diffusion kinetics of a lipophilic molecule across rabbit cornea.

Science.gov (United States)

Gupta, Chhavi; Chauhan, Anuj; Mutharasan, Raj; Srinivas, Sangly P

2010-04-01

To develop a kinetic model for representing the diffusion and partitioning of Rhodamine B (RhB), a fluorescent lipophilic molecule, across the cornea for gaining insights into pharmacokinetics of topical drugs to the eye. Rabbit corneas mounted underneath a custom-built scanning microfluorometer were perfused with Ringers on both sides of the tissue. After a step change in RhB on the tear side, transients of trans-corneal fluorescence of RhB were measured at a depth resolution approximately 8 microm. RhB distribution exhibited discontinuities at the interface between epithelium and stroma, and between stroma and endothelium. In each of the layers, fluorescence was non-uniform. Fluorescence was elevated in the epithelium and endothelium relative to the stroma. Modeling of RhB transport by diffusion in each layer and stipulation of partitioning of RhB at the cellular interfaces were required to account for trans-corneal penetration kinetics of RhB. The model parameters, estimated using the unsteady state trans-corneal RhB profiles, were found to be sensitive, and the model predicted the experimental profiles accurately. Conventional pharmacokinetic models that depict cornea as a single compartment do not predict the depth-dependent kinetics of RhB penetration. The proposed model incorporates realistic transport mechanisms and thereby highlights the influence of physicochemical properties of drugs on trans-corneal kinetics.

Influence of the He-Ne laser in different irradiation regimes on the cornea cells after ionizing irradiation

International Nuclear Information System (INIS)

Bulyakova, N.V.

1984-01-01

A study was made on epithelium cells of the eye cornea in mices entering the first postradiation mitosis. The influence of laser rays acting in the course of each exposure during one and the same period of time, or continuously, or by pulses was compared. The effect of laser radiation was evaluated from the frequency of occurrence of cells with chromosomal aberrations, mitotic index, coefficient of mitosis phases. It has been established that pulse radiation of a helium-neon laser in a certain operation regime produces a sanitating effect on cells of eye cornea epithelium in white mice damaged by ionizing radiation. Cells being killed in the interphase were not revealed in epithelium of the eye cornea. It enables to conclude that the amount of aberrant mitoses decreases as a result of postradiation repair of cells damaged by ionizing radiation, but not as a result of their elimination

A short-term study of corneal collagen cross-linking with hypo-osmolar riboflavin solution in keratoconic corneas

Directory of Open Access Journals (Sweden)

Shao-Feng Gu

2015-02-01

Full Text Available AIM: To report the 3mo outcomes of collagen cross-linking (CXL with a hypo-osmolar riboflavin in thin corneas with the thinnest thickness less than 400 μm without epithelium. METHODS: Eight eyes in 6 patients with age 26.2±4.8y were included in the study. All patients underwent CXL using a hypo-osmolar riboflavin solution after its de-epithelization. Best corrected visual acuity, manifest refraction, the thinnest corneal thickness, and endothelial cell density were evaluated before and 3mo after the procedure. RESULTS: The mean thinnest thickness of the cornea was 408.5±29.0 μm before treatment and reduced to 369.8±24.8 μm after the removal of epithelium. With the application of the hypo-osmolar riboflavin solution, the thickness increased to 445.0±26.5 μm before CXL and recover to 412.5±22.7 μm at 3mo after treatment, P=0.659. Before surgery, the mean K-value of the apex of the keratoconus corneas was 57.6±4.0 diopters, and slightly decreased (54.7±4.9 diopters after surgery (P=0.085. Mean best-corrected visual acuity was 0.55±0.23 logarithm of the minimal angle of resolution, and increased to 0.53±0.26 logarithm after surgery (P=0.879. The endothelial cell density was 2706.4±201.6 cells/mm2 before treatment, and slightly decreased (2641.2±218.2 cells/mm2 at last fellow up (P=0.002. CONCLUSION: Corneal collagen cross-linking with a hypo-osmolar riboflavin in thin corneas seems to be a promising treatment. Further study should be done to evaluate the safety and efficiency of CXL in thin corneas for the long-term.

The kinetics of interaction of porcine - alpha-, and porcine - beta -trypsin with intact and modified soybean trypsin inhibitor (kunitz)

International Nuclear Information System (INIS)

Hamid, M.A.

1994-01-01

The association of porcine trypsin with soybean trypsin inhibitor (Kunitz) resulted in characteristic changes in absorption spectrum, indicating an alteration of the micro environments of the enzyme chromophores as a consequence of the interaction. The rates of formation of the stable trypsin - inhibitor complexes from porcine - alpha - trypsin and soybean trypsin inhibitor and from porcine - beta - trypsin and either intact or modified soybean trypsin inhibitor were measured by mixing the equimolar concentration of the reactants in a Stopped - Flow apparatus at pH (4.5 to 10.0). The reaction of trypsin with soybean trypsin inhibitor was of first order with respect to the concentration of the reactants used. The rates of dissociation of the stable complexes, alpha - trypsin - soybean trypsin inhibitor, beta -trypsin - soybean trypsin inhibitor and beta -trypsin modified soybean trypsin inhibitor were also measured at pH (1.92 to 3.58). The values of first order rate constant, k/sub D/ obtained for the dissociation of all the three complexes were identical with one another. The kinetics results obtained for the porcine trypsin were compared with those of bovine trypsin system and it was suggested that the reaction mechanisms in both these systems were identical. (author)

Comparison of Scheimpflug imaging parameters between steep and keratoconic corneas of Caucasian eyes

Directory of Open Access Journals (Sweden)

Huseynova T

2016-04-01

Full Text Available Tukezban Huseynova,1 Farah Abdulaliyeva,2 Michele Lanza3 1Briz-L Eye Clinic, 2National Ophthalmology Center, Baku, Azerbaijan; 3Second University of Naples, Caserta, Campania, Italy Purpose: To compare the keratometric and pachymetric parameters of healthy eyes with those affected by steep cornea and keratoconus (KC using Scheimpflug camera.Setting: Briz-L Eye Clinic, Baku, Azerbaijan.Design: A cross-sectional study.Methods: In this study, 49 KC (Amsler–Krumeich stage 1 eyes and 36 healthy eyes were enrolled. A complete ophthalmic evaluation and a Scheimpflug camera scan were performed in every eye included in the study. Tomographic parameters such as parameters from the front and back cornea, maximum keratometry reading (Kmax, corneal volume (CV, anterior chamber volume (ChV, anterior chamber depth (ACD, anterior chamber angle (AC angle, keratometric power deviation (KPD, maximum front elevation (Max FE, and maximum back elevation (Max BE, as well as pachymetric progression indices (PPI, Ambrosio relational thickness (ART, index of surface variance (ISV, index of vertical asymmetry (IVA, center keratoconus index (CKI, index of height asymmetry (IHA, index of height decentration (IHD, and radius minimum (RM were collected and statistically compared between the two groups.Results: PPI, ART, ISV, IVA, CKI, IHA, IHD, and RM parameter values were significantly different (P<0.05 between the KC and healthy eyes. There were no significant differences in K mean and Q values of the frontal corneal parameters, as well as in Kmax, AC angle, RM, back, and front astigmatism, between stage 1 keratoconic and normal Caucasian eyes with steep cornea. All other parameters such as K mean and Q values of the back corneal parameters, Max FE, Max BE, ACD, ChV, and CV showed significant differences between the groups (P<0.05 for all. Conclusion: Scheimpflug imaging is able to detect corneal morphological differences between stage 1 KC eyes and healthy eyes with

Bacteriospermia in extended porcine semen.

Science.gov (United States)

Althouse, Gary C; Lu, Kristina G

2005-01-15

Bacteriospermia is a frequent finding in freshly extended porcine semen and can result in detrimental effects on semen quality and longevity if left uncontrolled. The primary source of bacterial contamination is the boar. Other sources that have been identified include environment, personnel, and the water used for extender preparation. A 1-year retrospective study was performed on submissions of extended porcine semen for routine quality control bacteriological screening at the University of Pennsylvania. Out of 250 sample submissions, 78 (31.2%) tested positive for bacterial contamination. The most popular contaminants included Enterococcus spp. (20.5%), Stenotrophomonas maltophilia (15.4%), Alcaligenes xylosoxidans (10.3%), Serratia marcescens (10.3%), Acinetobacter lwoffi (7.7%), Escherichia coli (6.4%), Pseudomonas spp. (6.4%), and others (23.0%). Prudent individual hygiene, good overall sanitation, and regular monitoring can contribute greatly in controlling bacterial load. Strategies that incorporate temperature-dependent bacterial growth and hyperthermic augmentation of antimicrobial activity are valuable for effective control of susceptible bacterial loads. Aminoglycosides remain the most popular antimicrobial class used in porcine semen extenders, with beta-lactam and lincosamide use increasing. With the advent of more novel antimicrobial selection and semen extender compositions in swine, prudent application and understanding of in vitro pharmacodynamics are becoming paramount to industry success in the use of this breeding modality.

Preparation of pre-cut corneas from fresh donated whole globes for Descemet's stripping automated keratoplasty: 3-year results at the Central Eye Bank of Iran.

Science.gov (United States)

Kanavi, Mozhgan Rezaei; Javadi, Mohammad Ali; Javadi, Fatemeh; Chamani, Tahereh

2014-09-01

To describe the technique and the results of the preparation of pre-cut corneas for Descemet's stripping automated endothelial keratoplasty (DSAEK) during a 3-year period at the Central Eye Bank of Iran (CEBI). The method of preparation of pre-cut corneas from donated whole globes at the CEBI is described and the frequency and percentage of pre-cut corneas prepared for DSAEK, between April 2009 and March 2012, are specified. Moreover, post-operative reports are reviewed for any complaints about using pre-cut tissues for DSAEK. Out of the 1,518 donated whole globes appropriate for DSAEK, 1,478 (97.4 %) pre-cut corneas were successfully prepared. The method of preparation failed in 40 (2.6 %) cases. Based on the eye bank post-operative reports, thickness of pre-cut tissues for DSAEK was deemed unacceptable in only 6 (0.4 %) cases prior to surgery; five of these were too thick and one was too thin. Preparation of pre-cut corneas, for DSAEK from donated whole globes, in the CEBI is a safe and easy method, with very good preservation of endothelial cells after the preparation of the pre-cut corneas and reduced risks from corneal manipulation.

Visual acuity outcomes in eyes with flat corneas after PRK.

Science.gov (United States)

Varssano, David; Waisbourd, Michael; Minkev, Liza; Sela, Tzahi; Neudorfer, Meira; Binder, Perry S

2013-06-01

To evaluate the impact of corneal curvatures less than 35 diopters (D) after photorefractive keratectomy (PRK) on visual acuity outcomes. Visual acuity outcomes of 5,410 eyes that underwent PRK from January 2006 to November 2010 were retrospectively analyzed for the impact of postoperative corneal curvatures on visual outcomes. All procedures were performed on a single platform (Allegretto 200Hz excimer laser; Alcon Laboratories, Inc., Irvine, CA). Main outcome measures were postoperative corrected distance visual acuity (CDVA) and loss of CDVA. Corneas with a measured or a calculated postoperative flat meridian less than 35 D and those with a measured postoperative steep meridian less than 35 D had worse postoperative CDVA than corneas with meridians of either 35 D or more (P ≤ .021). However, the preoperative CDVA was worse in the flatter curvatures in all comparisons performed (P ≤ .024). Consequently, the measured or calculated meridian curvature had no effect on CDVA loss (P ≥ .074). Postoperative corneal keratometry values (flat and steep meridians) less than 35 D did not have a predictive effect on the risk of losing visual acuity following myopic PRK performed on the Allegretto 200Hz excimer laser. Copyright 2013, SLACK Incorporated.

Effect of In Vitro Transcorneal Approach of Aceclofenac Eye Drops through Excised Goat, Sheep, and Buffalo Corneas

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Vivek Dave

2015-01-01

Full Text Available The current study involves the evaluation of factors that influence the transcorneal permeation of aqueous drops of aceclofenac ophthalmic formulation through freshly excised goat, sheep, and buffalo corneas. Aceclofenac formulation with different concentrations 0.1–0.5% (w/v and with different pH and different preservatives, was taken into account. The amount of drug permeated from different formulations was estimated using an Franz diffusion cell. A linear increase in drug permeation was observed with increase in pH (5.5 to 7.4. The apparent permeability coefficient was found to be maximum 15.01±0.45 on goat cornea and maximum transport of aceclofenac was observed at physiological pH of tears (i.e., 7. The results advocate that aceclofenac 0.5% (w/v ophthalmic solution (pH 7.0 containing BAK (0.01% provides maximum in vitro ocular permeability through goat, sheep, and buffalo corneas.

Removal of a barbed fish hook from the cornea of an 8-year-old boy.

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Kemp, Pavlina S; Shah, Ankoor S; Elliott, Alexandra T; Wan, Michael J

2017-10-01

This video article shows the surgical removal of a three-pronged barbed fish hook from the cornea and anterior chamber of an 8-year-old boy. The advance-and-cut method is shown: the embedded barbed point of the fish hook was advanced through the cornea, its point was cut off, and the shank of the hook was withdrawn. This method was chosen due to the three-pronged configuration of the fish hook to minimize additional trauma to the eye. Other methods of fish hook removal are briefly reviewed. Copyright © 2017 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.

Porcine UCHL1: genomic organization, chromosome localization and expression analysis

DEFF Research Database (Denmark)

Larsen, Knud; Madsen, Lone Bruhn; Bendixen, Christian

2012-01-01

to and protection from Parkinson’s disease. Here we report cloning, characterization, expression analysis and mapping of porcine UCHL1. The UCHL1 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine cDNA codes...... in developing porcine embryos. UCHL1 transcript was detected as early as 40 days of gestation. A significant decrease in UCHL1 transcript was detected in basal ganglia from day 60 to day 115 of gestation...

The ultrastructural alterations in rat corneas with experimentally-induced diabetes mellitus

International Nuclear Information System (INIS)

Take, G.; Karabay, G.; Erdogan, D.; Duyar, I.

2006-01-01

To examine the ultrastructural changes of rat corneas in streptozotocin (STZ) induced diabetes mellitus and the and the follow-up insulin treatment. Sprague-Dawley type rats were used for experimental procedures during the period from January to April 2003 at Baskent University, Ankara, Turkey. Rats were studied in four groups: group 1: controls, group 2 sham controls (single dose IV sodium citrate); group 3 STZ-induced diabetes mellitus (Single dose 45mg/kg STZ intravenously), group 4: diabetes mellitus + insulin treatment (8U/day). We observed degenerative changes in the epithelial layer, stromal keratocytes and endothelial cells in diabetic group. In contrast, the corneal layers have revealed positive alterations in the insulin-treated group. The statistical analysis, showed significant narrowing in the epithelial layer in the diabetic group (p0.02), whereas thickening was observed in the epithelial basement membrane and Descemet's membrane (p=0.002). It was determined that that diabetes mellitus causes degenerative changes in cornea, which are positively influenced by short-term insulin treatment. (author)

New stepwise cooling system for short-term porcine islet preservation.

Science.gov (United States)

Ikemoto, Tetsuya; Noguchi, Hirofumi; Fujita, Yasutaka; Takita, Morihito; Shimoda, Masayuki; Sugimoto, Koji; Jackson, Andrew; Naziruddin, Bashoo; Shimada, Mitsuo; Levy, Marlon F; Matsumoto, Shinichi

2010-10-01

Porcine islets are the most suitable for xeno-islet transplantation. However, it is necessary to establish an effective preservation method against its fragility. Recently, we developed a new cooling and preservation (Keep and Fresh [KFC]; FUJIYA Co, Tokushima, Japan) system, which can maintain viability of hepatocyte. In this study, we examined the KFC for porcine islet preservation. Isolated porcine islets were preserved in CMRL 1066 culture media with bovine serum at 37°C, 22°C, and 4°C and KFC for 24, 48, and 72 hours. Islet recovery rate, purity, and viability were evaluated. After 24-hour preservation, the recovery rate was the highest in the KFC, but no significant difference was found. After 48-hour preservation, the recovery rate by the KFC was 73.9% ± 17.3%, which was significantly higher than the other groups (48.7% ± 28.6% at 37°C, P KFC group, purities and viabilities were the highest among the groups after 24-, 48-, and 72-hour preservation. The KFC system significantly improved porcine islet preservation; therefore, the KFC might be useful for porcine islet preservation.

[Evaluation of Iris Morphology Viewed through Stromal Edematous Corneas by Infrared Camera].

Science.gov (United States)

Kobayashi, Masaaki; Morishige, Naoyuki; Morita, Yukiko; Yamada, Naoyuki; Kobayashi, Motomi; Sonoda, Koh-Hei

2016-02-01

We reported that the application of infrared camera enables us to observe iris morphology in Peters' anomaly through edematous corneas. To observe the iris morphology in bullous keratopathy or failure grafts with an infrared camera. Eleven bullous keratopathy or failure grafts subjects (6 men and 5 women, mean age ± SD; 72.7 ± 13.0 years old) were enrolled in this study. The iris morphology was observed by applying visible light mode and near infrared light mode of infrared camera (MeibomPen). The detectability of pupil shapes, iris patterns and presence of iridectomy was evaluated. Infrared mode observation enabled us to detect the pupil shapes in 11 out of 11 cases, iris patterns in 3 out of 11 cases, and presence of iridetomy in 9 out of 11 cases although visible light mode observation could not detect any iris morphological changes. Applying infrared optics was valuable for observation of the iris morphology through stromal edematous corneas.

Extraction Socket Preservation Using Porcine-Derived Collagen Membrane Alone or Associated with Porcine-Derived Bone. Clinical Results of Randomized Controlled Study

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Renzo Guarnieri

2017-03-01

Full Text Available Objectives: The aim of present randomized controlled clinical trial was to clinically evaluate hard tissue changes after extraction socket preservation procedures compared to natural spontaneous healing. Material and Methods: Thirty patients were enrolled in the present study and underwent single-tooth extraction in the premolar/molar areas. Ten sites were grafted with porcine-derived bone covered by collagen membrane, 10 covered by porcine-derived collagen membrane alone, and 10 underwent natural spontaneous healing. Vertical and horizontal bone changes after 3-month were evaluated at implant placement. Results: The vertical and horizontal bone changes at the extraction sockets treated with collagen membrane alone (vertical: -0.55 [SD 0.11] mm, and horizontal: -1.21 [SD 0.69] mm and collagen membrane plus porcine-derived bone (vertical: -0.37 [SD 0.7] mm, and horizontal: -0.91 [SD 0.53] mm were found significantly lower (P < 0.001, when compared to non-grafted sockets (vertical: -2.09 [SD 0.19] mm, and horizontal: -3.96 [SD 0.87] mm. In type 1 extraction sockets, in premolar sites, and in presence of vestibular bone thicknesses ≥ 1.5 mm, the use of collagen membrane alone revealed similar outcomes to those with additional graft material. Conclusions: At the re-entry surgery, extraction sockets grafted with porcine-derived bone and covered by collagen membrane, and extraction sockets covered by porcine-derived collagen membrane alone, showed significantly lower vertical and horizontal bone changes, compared to extraction sockets sites underwent natural spontaneous healing. However, a complete prevention of remodelling is not achievable, irrespective of the technique used.

Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

Science.gov (United States)

van Essen, T H; van Zijl, L; Possemiers, T; Mulder, A A; Zwart, S J; Chou, C-H; Lin, C C; Lai, H J; Luyten, G P M; Tassignon, M J; Zakaria, N; El Ghalbzouri, A; Jager, M J

2016-03-01

To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of the Viscoelastic Properties of the Human Cornea Using Scheimpflug Imaging in Inflation Experiment of Eye Globes

Science.gov (United States)

Lombardo, Giuseppe; Serrao, Sebastiano; Rosati, Marianna; Lombardo, Marco

2014-01-01

Purpose To demonstrate a Scheimpflug-based imaging procedure for investigating the depth- and time-dependent strain response of the human cornea to inflation testing of whole eye globes. Methods Six specimens, three of which with intact corneal epithelium, were mounted in a customized apparatus within a humidity and temperature-monitored wet chamber. Each specimen was subjected to two mechanical tests in order to measure corneal strain resulting from application of cyclic (cyclic regimen) and constant (creep regimen) stress by changing the intra-ocular pressure (IOP) within physiological ranges (18–42 mmHg). Corneal shape changes were analyzed as a function of IOP and both corneal stress-strain curves and creep curves were generated. Results The procedure was highly accurate and repeatable. Upon cyclic stress application, a biomechanical corneal elasticity gradient was found in the front-back direction. The average Young's modulus of the anterior cornea ranged between 2.28±0.87 MPa and 3.30±0.90 MPa in specimens with and without intact epithelium (P = 0.05) respectively. The Young's modulus of the posterior cornea was on average 0.21±0.09 MPa and 0.17±0.06 MPa (P>0.05) respectively. The time-dependent strain response of the cornea to creep testing was quantified by fitting data to a modified Zener model for extracting both the relaxation time and compliance function. Conclusion Cyclic and creep mechanical tests are valuable for investigating the strain response of the intact human cornea within physiological IOP ranges, providing meaningful results that can be translated to clinic. The presence of epithelium influences the results of anterior corneal shape changes when monitoring deformation via Scheimpflug imaging in inflation experiments of whole eye globes. PMID:25397674

Fibre optic spectrophotometry for the in vitro evaluation of ultraviolet radiation (UVR) spectral transmittance of rabbit corneas

International Nuclear Information System (INIS)

Walsh, J E; Fleming, D P; Bergmanson, J P G; Koehler, L V; Doughty, M J; Harmey, J H

2008-01-01

A fibre optic spectrophotometer front-end system for measuring corneas to overcome shortcomings associated with existing instruments was tested. The system allowed prompt measurement postmortem, minimizing beam pathlength to reduce the effects of scatter and unwanted refraction and eliminated optical interfaces and cuvette media. Rabbit corneas were excised immediately postmortem and placed on a detecting fibre optic coupled to an Ocean Optics spectrophotometer and illuminated by a deuterium–halogen source. The compact instrument with its small beam size allowed tissue profiling at test points across the corneal surface and efficient interchange for comparison of different tissues. This simplified system operation allowed rapid tissue altering to study induced changes on transmittance. The corneal transmittance data showed a consistent sharp cut-off at 320 nm in the ultraviolet radiation (UVR) spectrum, which decayed rapidly from postmortem swelling. Inter- and intra-corneal consistency was demonstrated by comparing data from different regions of the same cornea and those from opposite eyes. Changes to the spectra, particularly in the UVB below 300 nm, were evident when the corneal epithelium was removed, indicating that this layer is not the only corneal UVR filter. The new system reduced much of the variability associated with previous methods, as it rapidly measured corneal transmittance postmortem. Data are in broad agreement with published transmittance curves. The removal of the corneal epithelium revealed a substantial stromal contribution to the overall corneal UVR absorption, suggesting that corneas with pathologically or iatrogenically thinned stromas are less effective UVR blockers

Netrin-1 regulates fibrocyte accumulation in the decellularized fibrotic scleroderma lung microenvironment and in bleomycin induced pulmonary fibrosis

Science.gov (United States)

Sun, Huanxing; Zhu, Yangyang; Pan, Hongyi; Chen, Xiaosong; Balestrini, Jenna L.; Lam, TuKiet T.; Kanyo, Jean E.; Eichmann, Anne; Gulati, Mridu; Fares, Wassim H.; Bai, Hanwen; Feghali-Bostwick, Carol A.; Gan, Ye; Peng, Xueyan; Moore, Meagan W.; White, Eric S.; Sava, Parid; Gonzalez, Anjelica L.; Cheng, Yuwei; Niklason, Laura E.; Herzog, Erica L.

2017-01-01

Objectives Fibrocytes are collagen-producing leukocytes that accumulate in Scleroderma-associated interstitial lung disease (SSc-ILD) via unknown mechanisms. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in Scleroderma has not been explored. This study uses a novel translational platform based on decellularized human lungs to determine whether the scleroderma lung ECM controls fibrocyte development from peripheral blood mononuclear cells. Methods Decellularized scaffolds prepared from healthy and fibrotic Scleroderma lung explants underwent biomechanical evaluation using tensile testing and biochemical analysis using proteomics. Cells from healthy and SSc-ILD subjects were cultured on these scaffolds, and CD45+Pro-ColIα1+ cells meeting criteria for fibrocytes were quantified. The contribution of Netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and via the inhalational administration of bleomycin to Netrin-1+/− mice. Results Compared to control lung scaffold, SSc-ILD lung scaffolds showed aberrant anatomy, enhanced stiffness, and abnormal extracellular matrix composition. Culture of control cells in Scleroderma scaffolds increased Pro-ColIα1+ production, which was stimulated by enhanced stiffness and abnormal ECM composition. SSc-ILD cells demonstrated increased Pro-ColIα1 responsiveness to Scleroderma lung scaffolds, but not enhanced stiffness. Enhanced Netrin-1 expression was seen on CD14lo SSc-ILD cells and antibody mediated Netrin-1 neutralization attenuated CD45+Pro-ColIα1+ detection in all settings. Netrin-1+/− mice were protected from bleomycin induced lung fibrosis and fibrocyte accumulation. Conclusion Factors present in Scleroderma lung matrices regulate fibrocyte accumulation via a Netrin-1-dependent pathway. Netrin-1 regulates bleomycin induced murine pulmonary fibrosis. Netrin-1 might be a novel therapeutic target in SSc-ILD. PMID:26749424

Porcine embryonic stem cells

DEFF Research Database (Denmark)

Hall, Vanessa Jane

2008-01-01

The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

3D refraction correction and extraction of clinical parameters from spectral domain optical coherence tomography of the cornea.

Science.gov (United States)

Zhao, Mingtao; Kuo, Anthony N; Izatt, Joseph A

2010-04-26

Capable of three-dimensional imaging of the cornea with micrometer-scale resolution, spectral domain-optical coherence tomography (SDOCT) offers potential advantages over Placido ring and Scheimpflug photography based systems for accurate extraction of quantitative keratometric parameters. In this work, an SDOCT scanning protocol and motion correction algorithm were implemented to minimize the effects of patient motion during data acquisition. Procedures are described for correction of image data artifacts resulting from 3D refraction of SDOCT light in the cornea and from non-idealities of the scanning system geometry performed as a pre-requisite for accurate parameter extraction. Zernike polynomial 3D reconstruction and a recursive half searching algorithm (RHSA) were implemented to extract clinical keratometric parameters including anterior and posterior radii of curvature, central cornea optical power, central corneal thickness, and thickness maps of the cornea. Accuracy and repeatability of the extracted parameters obtained using a commercial 859nm SDOCT retinal imaging system with a corneal adapter were assessed using a rigid gas permeable (RGP) contact lens as a phantom target. Extraction of these parameters was performed in vivo in 3 patients and compared to commercial Placido topography and Scheimpflug photography systems. The repeatability of SDOCT central corneal power measured in vivo was 0.18 Diopters, and the difference observed between the systems averaged 0.1 Diopters between SDOCT and Scheimpflug photography, and 0.6 Diopters between SDOCT and Placido topography.

The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.

Science.gov (United States)

Couture, Camille; Zaniolo, Karine; Carrier, Patrick; Lake, Jennifer; Patenaude, Julien; Germain, Lucie; Guérin, Sylvain L

2016-02-01

Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Successful treatment of Fusarium keratitis with cornea transplantation and topical and systemic voriconazole.

NARCIS (Netherlands)

Klont, R.R.; Eggink, C.A.; Rijs, A.J.M.M.; Wesseling, P.; Verweij, P.E.

2005-01-01

A case of invasive Fusarium keratitis in a previously healthy male patient was treated successfully with cornea transplantation and systemic and topical voriconazole after treatment failure with topical amphotericin B and systemic itraconazole. Topical voriconazole was well tolerated, and, in

A Raman scattering and FT-IR spectroscopic study on the effect of the solar radiation in Antarctica on bovine cornea

Science.gov (United States)

Yamamoto, Tatsuyuki; Murakami, Naoki; Yoshikiyo, Keisuke; Takahashi, Tetsuya; Yamamoto, Naoyuki

2010-01-01

The Raman scattering and FT-IR spectra of the corneas, transported to the Syowa station in Antarctica and exposed to the solar radiation of the mid-summer for four weeks, were studied to reveal that type IV collagen involved in corneas were fragmented. The amide I and III Raman bands were observed at 1660 and 1245 cm -1, respectively, and the amide I and II infrared bands were observed at 1655 and 1545 cm -1, respectively, for original corneas before exposure. The background of Raman signals prominently increased and the ratio of amide II infrared band versus amide I decreased by the solar radiation in Antarctica. The control experiment using an artificial UV lamp was also performed in laboratory. The decline rate of the amide II/amide I was utilized for estimating the degree of fragmentation of collagen, to reveal that the addition of vitamin C suppressed the reaction while the addition of sugars promoted it. The effect of the solar radiation in Antarctica on the corneas was estimated as the same as the artificial UV lamp of four weeks (Raman) or one week (FT-IR) exposure.

The Effect of Systemic Amantadine Sulfate on Malondialdehyde and Total Thiol Levels in Rat Corneas

Directory of Open Access Journals (Sweden)

Züleyha Yalniz-Akkaya

2014-01-01

Full Text Available Purpose: To evaluate the malondialdehyde (MDA and total thiol (sulfhydryl, SH levels in rat corneas after intraperitoneal injection of amantadine sulfate. Methods: A total of 12 Wistar albino rats were divided into two groups: control group (n = 6 and amantadine group (n = 6. Balanced salt solution (1 mL, 0.9% NaCl, twice/day was injected into rats in control group. Amantadine sulfate (2 mg/1 mL, twice/day was injected into rats in amantadine group. In each group, two rats were injected for 1 week, two received injections for 1 month, and two rats received injections for 3 months. The corneas were homogenized and MDA and SH levels were measured spectroflourometrically. Results: In control group, median MDA and SH levels were 2.37 (range, 0.92-3.60 and 25.35 (range, 6.30-54.0 nmol/mg, respectively. In amantadine group, median MDA and SH levels were 3.57 (range, 1.25-5.92 and 32.65 (range, 3.30-48.3 nmol/mg, respectively. The difference between this two groups regarding MDA (P = 0.14 and SH (P = 1.0 levels was statistically insignificant. Conclusion: Systemically administered amantadine sulfate seems not to cause MDA and SH imbalance in rat corneas.

Rocking media over ex vivo corneas improves this model and allows the study of the effect of proinflammatory cytokines on wound healing.

Science.gov (United States)

Deshpande, Pallavi; Ortega, Ílida; Sefat, Farshid; Sangwan, Virender S; Green, Nicola; Claeyssens, Frederik; MacNeil, Sheila

2015-02-05

The aim of this work was to develop an in vitro cornea model to study the effect of proinflammatory cytokines on wound healing. Initial studies investigated how to maintain the ex vivo models for up to 4 weeks without loss of epithelium. To study the effect of cytokines, corneas were cultured with the interleukins IL-17A, IL-22, or a combination of IL-17A and IL-22, or lipopolysaccharide (LPS). The effect of IL-17A on wound healing was then examined. With static culture conditions, organ cultures deteriorated within 2 weeks. With gentle rocking of media over the corneas and carbon dioxide perfusion, the ex vivo models survived for up to 4 weeks without loss of epithelium. The cytokine that caused the most damage to the cornea was IL-17A. Under static conditions, wound healing of the central corneal epithelium occurred within 9 days, but only a single-layered epithelium formed whether the cornea was exposed to IL-17A or not. With rocking of media gently over the corneas, a multilayered epithelium was achieved 9 days after wounding. In the presence of IL-17A, however, there was no wound healing evident. Characterization of the cells showed that wherever epithelium was present, both differentiated cells and highly proliferative cells were present. We propose that introducing rocking to extend the effective working life of this model and the introduction of IL-17A to this model to induce aspects of inflammation extend its usefulness to study the effects of agents that influence corneal regeneration under normal and inflamed conditions. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

Brittle Cornea Syndrome Associated with a Missense Mutation in the Zinc-Finger 469 Gene

DEFF Research Database (Denmark)

Christensen, Anne Elisabeth; Knappskog, Per Morten; Midtbø, Marit

2010-01-01

Purpose: To investigate the diverse clinical manifestations, identify the causative mutation and explain the association with red hair in a family with brittle cornea syndrome (BCS). Methods: Eight family members in three generations underwent ophthalmic, dental, and general medical examination...... mapping with SNP markers, DNA sequencing, and MC1R genotyping. Results: At 42 and 48 years of age, respectively, both affected individuals were blind due to retinal detachment and secondary glaucoma. They had extremely thin and bulging corneas, velvety skin, chestnut colored hair, scoliosis, reduced BMD......, dental anomalies, hearing loss and minor cardiac defects. The morphologies of the skin biopsies were normal except that in some areas slightly thinner collagen fibrils were seen in one of the affected individuals. Molecular genetic analysis revealed a novel missense mutation of ZNF469, c.10016G...

Isolation and purification of porcine LH for radioimmunoassay and radioreceptor assay

International Nuclear Information System (INIS)

Ziecik, A.; Goralska, M.; Krzymowski, T.; Pogorzelski, K.

1979-01-01

The procedure of isolation and purification of LH from porcine pituitary glands is described. From 1 kg of pituitary glands 150 mg of LH GPZ-1 preparation of high purity were obtained. Immunization of rabbits with the prepared hormone gave homogeneous antibodies against porcine LH with high affinity and low cross-reactions with FSH. Radioreceptor assay with the use of the prepared porcine LH demonstrated the high capacity of cell membrane receptors of the boar tests for binding this hormone. (author)

Immunological half-life of porcine proinsulin C-peptide

Energy Technology Data Exchange (ETDEWEB)

Oyama, H; Horino, M; Matsumura, S [Kawasaki Medical Coll., Kurashiki (Japan). Div. of Endocrinology; Kobayshi, K; Suetsugu, N [Yamaguchi Univ., Ube (Japan). School of Medicine

1975-11-01

Immunological half-lifes of injected porcine C-peptide and insulin with RIA were studied and calculated as 9.8 and 8.0 minutes. Higher circulating levels of C-peptide as compared to insulin in normal young swines lead to speculation about a longer half-life of C-peptide. This hypothesis was verified in this study. Immunological half-lifes of porcine proinsulin and insulin in the pig were 20 and 6 minutes, respectively.

Clinical and epidemiological aspects of cornea transplant patients of a reference hospital.

Science.gov (United States)

Cruz, Giovanna Karinny Pereira; Azevedo, Isabelle Campos de; Carvalho, Diana Paula de Souza Rego Pinto; Vitor, Allyne Fortes; Santos, Viviane Euzébia Pereira; Ferreira, Marcos Antonio

2017-06-08

clinically characterizing cornea transplant patients and their distribution according to indicated and post-operative conditions of cornea transplantation, as well as estimating the average waiting time. a cross-sectional, descriptive and analytical study performed for all cornea transplants performed at a reference service (n=258). Data were analyzed using Statistical Package for the Social Sciences, version 20.0. the main indicator for cornea transplant was keratoconus. The mean waiting time for the transplant was approximately 5 months and 3 weeks for elective transplants and 9 days for urgent cases. An association between the type of corneal disorder with gender, age, previous surgery, eye classification, glaucoma and anterior graft failure were found. keratoconus was the main indicator for cornea transplant. Factors such as age, previous corneal graft failure (retransplantation), glaucoma, cases of surgeries prior to cornea transplant (especially cataract surgery) may be related to the onset corneal endothelium disorders. caracterizar clínicamente los pacientes trasplantados y su distribución, con descripción de las condiciones indicadoras y posoperatorias de los trasplantes de córneas, así como estimar el tiempo promedio en la fila de espera. estudio epidemiológico, transversal, descriptivo y analítico, realizado con todos los trasplantes de córnea realizados en un servicio de referencia (n=258). Los datos fueron analizados con el software Statistical Package for the Social Sciences, versión 20.0. la principal condición indicadora para el trasplante de córnea fue el queratocono. El tiempo promedio en fila de espera para realización del trasplante fue de aproximadamente 5 meses y tres semanas, para trasplantes electivos y de 9 días para los casos de urgencia. Existió asociación entre el tipo de disturbio de la córnea con: sexo, intervalo etario, cirugía previa, clasificación del ojo, glaucoma y rechazo del injerto anterior. el queratocono fue

Emerging technologies to create inducible and genetically defined porcine cancer models

Directory of Open Access Journals (Sweden)

Lawrence B Schook

2016-02-01

Full Text Available There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models.

Science.gov (United States)

Schook, Lawrence B; Rund, Laurie; Begnini, Karine R; Remião, Mariana H; Seixas, Fabiana K; Collares, Tiago

2016-01-01

There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic, and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae.

Science.gov (United States)

Turner, Helen C; Alvarez, Lawrence J; Candia, Oscar A; Bernstein, Audrey M

2003-10-01

To characterize the serotonin (5-HT) receptors linked to the modulation of adenylyl cyclase activity in rabbit, porcine and human conjunctivae. Serotonin receptor-subtype expression was examined using reverse transcription-polymerase chain reaction (RT-PCR) and receptor subtype-specific polyclonal antibodies for the immunofluorescent labeling of conjunctival cryosections. In addition, measurements of the effects of serotonergics on the short-circuit current (I(sc)) across rabbit and porcine conjunctivae were contrasted. RT-PCR assays indicated the expression of 5-HT(1B ) and 5-HT(1D) receptors, subtypes negatively coupled to adenylyl cyclase, in the rabbit conjunctiva. This approach also suggested the co-expression of 5-HT(1B), 5-HT(1D), 5-HT(1F), 5-HT(4) and 5-HT(7) mRNA's in the porcine conjunctiva, and 5-HT( 1D), 5-HT(1F) and 5-HT(7) in the human conjunctiva. Since the 5-HT(4) and 5-HT(7) receptors are positively linked to adenylyl cyclase, these results implied that the porcine and human tissues exhibited subtypes both positively and negatively linked to the enzyme. However, immunohistochemical observations, using currently available antibodies solely localized the 5-HT(7) moiety in the porcine and human epithelia, suggested that the 1B/1D forms may be minor elements. Consistent with this prospect, 5-HT was a stimulant of the transepithelial I(sc) across the porcine conjunctiva, an opposite response from earlier findings that demonstrated inhibitory effects by 5-HT on the rabbit I(sc), which are now explained by the localization of the 1B/1D receptors in the rabbit stratified epithelium. The 5-HT receptors expressed by mammalian conjunctivae are not identical. In terms of 5-HT receptor expression, the porcine tissue may be a more appropriate model for human, than is the rabbit, in that 5-HT may serve as a secretagogue in the human epithelium.

Cornea and anterior eye assessment with placido-disc keratoscopy, slit scanning evaluation topography and scheimpflug imaging tomography.

Science.gov (United States)

Martin, Raul

2018-03-01

Current corneal assessment technologies make the process of corneal evaluation extremely fast and simple and several devices and technologies allow to explore and to manage patients. The purpose of this special issue is to present and also to update in the evaluation of cornea and ocular surface and this second part, reviews a description of the corneal topography and tomography techniques, providing updated information of the clinical recommendations of these techniques in eye care practice. Placido-based topographers started an exciting anterior corneal surface analysis that allows the development of current corneal tomographers that provide a full three-dimensional reconstruction of the cornea including elevation, curvature, and pachymetry data of anterior and posterior corneal surfaces. Although, there is not an accepted reference standard technology for corneal topography description and it is not possible to determine which device produces the most accurate topographic measurements, placido-based topographers are a valuable technology to be used in primary eye care and corneal tomograhers expanding the possibilities to explore cornea and anterior eye facilitating diagnosis and follow-up in several situations, raising patient follow-up, and improving the knowledge regarding to the corneal anatomy. Main disadvantages of placido-based topographers include the absence of information about the posterior corneal surface and limited corneal surface coverage without data from the para-central and/or peripheral corneal surface. However, corneal tomographers show repeatable anterior and posterior corneal surfaces measurements, providing full corneal thickness data improving cornea, and anterior surface assessment. However, differences between devices suggest that they are not interchangeable in clinical practice.

Eye evolution: lens and cornea as an upgrade of animal visual system

Czech Academy of Sciences Publication Activity Database

Jonášová, Kristýna; Kozmik, Zbyněk

2008-01-01

Roč. 19, č. 2 (2008), s. 71-81 ISSN 1084-9521 R&D Projects: GA AV ČR IAA500520604; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50520514 Keywords : eye * lens * cornea Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.528, year: 2008

Pathways and Mechanisms Underlying the Photophysics and Photochemistry of Riboflavin induced cornea crosslinking

DEFF Research Database (Denmark)

Breitenbach, Thomas; Ogilby, Peter Remsen

In this talk, we will describe general pathways involved in the photophysics of a photosensitized process, which can lead to crosslinking due to light excitation of Riboflavin in the cornea. Furthermore, we will elucidate different aspects of reactions that can produce crosslinks, with respect...

[Characteristics of porcine thoracic arteries fixed with polyepoxy compound].

Science.gov (United States)

Yu, Xi-Xun; Chen, Huai-Qing

2005-09-01

To investigate the characteristics of porcine thoracic arteries fixed with ethylene glycol diglycidyl ether (EX-810) and to provide the proper scaffold materials for tissue-engineered blood vessel. The porcine thoracic arteries were respectively treated with 40 ml/L EX-810 and 6.25 g/L glutaraldehyde, and then they were examined with naked-eye, light microscope and scanning electron microscope. The fixation index determination, the amino acid analysis and the biomechanics test were also performed. The antigenicity of vascular tissues can be diminished by EX-810 through getting rid of cell in the vascular tissues or reducing the level of free amino groups in the vascular tissues. The structural integrity of vascular tissues can be preserved after treatment with EX-810. It was also found that the EX-810-fixed porcine vascular tissues appeared more similar to the natural vascular tissues in color and mechanical properties, and were more pliable than the glutaraldehyde-fixed tissues. The EX-810-fixed porcine thoracic arteries with low cytotoxicity and low antigenicity showed favorable characteristic similar to those of natural vessel, and it should be a promising material for fabricating scaffold of tissue-engineered blood vessel.

Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin

Indian Academy of Sciences (India)

Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. Qizhuang Lv Kangkang Guo Tao Wang ... Keywords. Cellular protein; FHC; ORF4 protein; porcine circovirus type 2 (PCV2); yeast two-hybrid ... Journal of Biosciences | News ...

Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine.

LENUS (Irish Health Repository)

Judge, Eoin P

2014-09-01

The porcine model has contributed significantly to biomedical research over many decades. The similar size and anatomy of pig and human organs make this model particularly beneficial for translational research in areas such as medical device development, therapeutics and xenotransplantation. In recent years, a major limitation with the porcine model was overcome with the successful generation of gene-targeted pigs and the publication of the pig genome. As a result, the role of this model is likely to become even more important. For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. An increasing number of lung conditions are being studied and modeled in the pig. Genetically modified porcine models of cystic fibrosis have been generated that, unlike mouse models, develop lung disease similar to human cystic fibrosis. However, the scientific literature relating specifically to porcine lung anatomy and airway histology is limited and is largely restricted to veterinary literature and textbooks. Furthermore, methods for in vivo lung procedures in the pig are rarely described. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

Preservation of enucleated porcine eyes for use in a wet laboratory

NARCIS (Netherlands)

Nibourg, Lisanne M.; Koopmans, Steven A.

PURPOSE: To design a method to preserve enucleated porcine eyes for use in a wet laboratory. SETTING: Laboratory of Experimental Ophthalmology, University Medical Center Groningen, the Netherlands. DESIGN: Experimental study. METHODS: Porcine eyes were preserved using 15 methods including salt

Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

Science.gov (United States)

Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

2015-01-01

Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry.

Possibilities of microscopic detection of isolated porcine proteins in model meat products

Directory of Open Access Journals (Sweden)

Michaela Petrášová

2016-05-01

Full Text Available In recent years, various protein additives intended for manufacture of meat products have increasing importance in the food industry. These ingredients include both, plant-origin as well as animal-origin proteins. Among animal proteins, blood plasma, milk protein or collagen are used most commonly. Collagen is obtained from pork, beef, and poultry or fish skin. Collagen does not contain all the essential amino acids, thus it is not a full protein in terms of essential amino acids supply for one's organism. However, it is rather rich in amino acids of glycine, hydroxyproline and proline which are almost absent in other proteins and their synthesis is very energy intensive. Collagen, which is added to the soft and small meat products in the form of isolated porcine protein, significantly affects the organoleptic properties of these products. This work focused on detection of isolated porcine protein in model meat products where detection of isolated porcine protein was verified by histological staining and light microscopy. Seven model meat products from poultry meat and 7 model meat products from beef and pork in the ratio of 1:1, which contained 2.5% concentration of various commercially produced isolated porcine proteins, were examined. These model meat products were histologically processed by means of cryosections and stained with hematoxylin-eosin staining, toluidine blue staining and Calleja. For the validation phase, Calleja was utilized. To determine the sensitivity and specificity, five model meat products containing the addition of isolated porcine protein and five model meat products free of it were used. The sensitivity was determined for isolated porcine protein at 1.00 and specificity was determined at 1.00. The detection limit of the method was at the level of 0.001% addition. Repeatability of the method was carried out using products with addition as well as without addition of isolated porcine protein and detection was repeated

Surgical induction of choroidal neovascularization in a porcine model

DEFF Research Database (Denmark)

Lassota, Nathan; Kiilgaard, Jens Folke; Prause, Jan Ulrik

2007-01-01

PURPOSE: To develop a reproducible surgical technique for the induction of choroidal neovascularization (CNV) in the subretinal space of porcine eyes and to analyse the resulting CNV clinically and histologically. METHODS: Two different modifications of a surgical technique previously described...... were compared with the original method. In ten porcine eyes retinal pigment epithelial (RPE) cells were removed using a silicone tipped cannula, in ten porcine eyes Bruch's membrane was perforated once with a retinal perforator without prior RPE removal and in ten eyes RPE removal was followed...... by a single perforation of Bruch's membrane. Fifteen of the eyes, five from each group, were enucleated 30 minutes after surgery, while the remaining eyes were enucleated after 14 days. Prior to enucleation, at day 14, fundus photographs and fluorescein angiograms were obtained. Eyes were examined by light...

[Current approaches to evaluating the anatomic and functional status of the cornea].

Science.gov (United States)

Avetisov, S E; Borodina, N V; Kobzova, M V; Musaeva, G M

2010-01-01

The review provides data on current methods for evaluating the anatomic and functional status of the cornea (light refraction, light transmission, and biomechanical properties, in particular). It analyzes the main advantages and disadvantages of basic (biomicroscopy, endothelial microscopy, ophthalmometry, topography, and pachymetry) and special (confocal microscopy, optical coherence tomography, ultrasound biomicroscopy, aberrometry, bidirectional corneal applanation, and keratoesthesiometry) studies.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

International Nuclear Information System (INIS)

Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

2016-01-01

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

Energy Technology Data Exchange (ETDEWEB)

Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Cui, Xiang-Shun; Kim, Nam-Hyung [Department of Animal Science, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Sun, Shao-Chen, E-mail: sunsc@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

2016-06-01

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

Science.gov (United States)

Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

2003-10-01

A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

Novel porcine repetitive elements

Directory of Open Access Journals (Sweden)

Nonneman Dan J

2006-12-01

Full Text Available Abstract Background Repetitive elements comprise ~45% of mammalian genomes and are increasingly known to impact genomic function by contributing to the genomic architecture, by direct regulation of gene expression and by affecting genomic size, diversity and evolution. The ubiquity and increasingly understood importance of repetitive elements contribute to the need to identify and annotate them. We set out to identify previously uncharacterized repetitive DNA in the porcine genome. Once found, we characterized the prevalence of these repeats in other mammals. Results We discovered 27 repetitive elements in 220 BACs covering 1% of the porcine genome (Comparative Vertebrate Sequencing Initiative; CVSI. These repeats varied in length from 55 to 1059 nucleotides. To estimate copy numbers, we went to an independent source of data, the BAC-end sequences (Wellcome Trust Sanger Institute, covering approximately 15% of the porcine genome. Copy numbers in BAC-ends were less than one hundred for 6 repeat elements, between 100 and 1000 for 16 and between 1,000 and 10,000 for 5. Several of the repeat elements were found in the bovine genome and we have identified two with orthologous sites, indicating that these elements were present in their common ancestor. None of the repeat elements were found in primate, rodent or dog genomes. We were unable to identify any of the replication machinery common to active transposable elements in these newly identified repeats. Conclusion The presence of both orthologous and non-orthologous sites indicates that some sites existed prior to speciation and some were generated later. The identification of low to moderate copy number repetitive DNA that is specific to artiodactyls will be critical in the assembly of livestock genomes and studies of comparative genomics.

Radiation sensitivity of bacteria and virus in porcine xenoskin for dressing agent

International Nuclear Information System (INIS)

Jo, Eu-Ri; Jung, Pil-Mun; Choi, Jong-il; Lee, Ju-Woon

2012-01-01

In this study, gamma irradiation sensitivities of bacteria and viruses in porcine skin were evaluated to establish the optimum sterilization condition for the dressing material and a xenoskin graft. Escherichia coli and Bacillus subtilis were used as model pathogens and inoculated at 10 6 –10 7 log CFU/g. As model viruses, porcine parvovirus (PPV), bovine viral diarrhea virus (BVDV), and poliovirus were used and inoculated at 10 5 –10 6 TCID 50 /g into porcine skin. The D 10 value of E. coli was found to be 0.25±0.1 kGy. B. subtilis endospores produced under stressful environmental conditions showed lower radiation sensitivity as D 10 was 3.88±0.3 kGy in porcine skin. The D 10 values of PPV, BVDV, and poliovirus were found to be 1.73±0.2, 3.81±0.2, and 6.88±0.3 kGy, respectively. These results can offer the basic information required for inactivating pathogens by gamma irradiation and achieving dressing material and porcine skin grafts.

Exploration of alternative test methods to evaluate phototoxicity of ophthalmic agents by using Statens Seruminstitut Rabbit Cornea cell lines and 3D human reconstituted cornea models.

Directory of Open Access Journals (Sweden)

Soyoung Kim

Full Text Available Many chemicals have been reported to induce phototoxicity. The absorbance of light energy within the sunlight range is a common characteristic of phototoxicity. The 3T3 NRU phototoxicity test (PT in 3T3 mouse skin fibroblasts has been used to identify the phototoxic potential induced by excited chemicals after exposure to ultra violet (UV. However, as phototoxicity may occur in ocular cells, it is necessary to develop a more suitable test for cornea-derived cells. In this study, we attempted to establish a new in vitro PT method in rabbit corneal cell lines (SIRC. We evaluated five ophthalmic agents, ciprofloxacin, levofloxacin, lomefloxacin, norfloxacin, and tetracycline, for their cytotoxic potential and in vitro phototoxicity. The results obtained using 3D human corneal models revealed that the UV-induced eye tissue toxicity by the test substances showed good correlation with those obtained using the in vitro phototoxicity test. However, the results from the 3D PT for ciprofloxacin, norfloxacin, and tetracycline in the 3D human cornea model were only partially comparable. Therefore, we suggest the SIRC cell line as a new phototoxicity test model; however, a sequential testing strategy, such as 3D PT, was also proposed to obtain relevant information for topical eye agents.

Transcriptome Profiling of Neovascularized Corneas Reveals miR-204 as a Multi-target Biotherapy Deliverable by rAAVs

Directory of Open Access Journals (Sweden)

Yi Lu

2018-03-01

Full Text Available Corneal neovascularization (NV is the major sight-threatening pathology caused by angiogenic stimuli. Current drugs that directly target pro-angiogenic factors to inhibit or reverse the disease require multiple rounds of administration and have limited efficacies. Here, we identify potential anti-angiogenic corneal microRNAs (miRNAs and demonstrate a framework that employs discovered miRNAs as biotherapies deliverable by recombinant adeno-associated viruses (rAAVs. By querying differentially expressed miRNAs in neovascularized mouse corneas induced by alkali burn, we have revealed 39 miRNAs that are predicted to target more than 5,500 differentially expressed corneal mRNAs. Among these, we selected miR-204 and assessed its efficacy and therapeutic benefit for treating injured corneas. Our results show that delivery of miR-204 by rAAV normalizes multiple novel target genes and biological pathways to attenuate vascularization of injured mouse cornea. Importantly, this gene therapy treatment alternative is efficacious and safe for mitigating corneal NV. Overall, our work demonstrates the discovery of potential therapeutic miRNAs in corneal disorders and their translation into viable treatment alternatives.

Sequence and expression analyses of porcine ISG15 and ISG43 genes.

Science.gov (United States)

Huang, Jiangnan; Zhao, Shuhong; Zhu, Mengjin; Wu, Zhenfang; Yu, Mei

2009-08-01

The coding sequences of porcine interferon-stimulated gene 15 (ISG15) and the interferon-stimulated gene (ISG43) were cloned from swine spleen mRNA. The amino acid sequences deduced from porcine ISG15 and ISG43 genes coding sequence shared 24-75% and 29-83% similarity with ISG15s and ISG43s from other vertebrates, respectively. Structural analyses revealed that porcine ISG15 comprises two ubiquitin homologues motifs (UBQ) domain and a conserved C-terminal LRLRGG conjugating motif. Porcine ISG43 contains an ubiquitin-processing proteases-like domain. Phylogenetic analyses showed that porcine ISG15 and ISG43 were mostly related to rat ISG15 and cattle ISG43, respectively. Using quantitative real-time PCR assay, significant increased expression levels of porcine ISG15 and ISG43 genes were detected in porcine kidney endothelial cells (PK15) cells treated with poly I:C. We also observed the enhanced mRNA expression of three members of dsRNA pattern-recognition receptors (PRR), TLR3, DDX58 and IFIH1, which have been reported to act as critical receptors in inducing the mRNA expression of ISG15 and ISG43 genes. However, we did not detect any induced mRNA expression of IFNalpha and IFNbeta, suggesting that transcriptional activations of ISG15 and ISG43 were mediated through IFN-independent signaling pathway in the poly I:C treated PK15 cells. Association analyses in a Landrace pig population revealed that ISG15 c.347T>C (BstUI) polymorphism and the ISG43 c.953T>G (BccI) polymorphism were significantly associated with hematological parameters and immune-related traits.

Public Health Implications of the 1540 nm Laser on the Cornea

Science.gov (United States)

2007-05-30

of Public Health Degree 30 May 2007 - Thomas J01vfson, Ph.D. Department of Preventive Medicine & Biometrics Committee Member Christine Kasper, ’,D...Xu and Alfano 2005) This absorption characteristic is useful in refractive surgery of the eye because the cornea is approximately 80% water...retina. Invest Ophthalmol Vis Sci 35: 3032-44; 1994. Xu M, Alfano RR. Fractal mechanisms of light scattering in biological tissue and cells. Opt

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

Science.gov (United States)

2013-03-01

aSMA ) synthesis. Second, we proposed to develop an advanced ex vivo organ culture system using viable explants of rabbit corneas, and assess the...effect of the most effective triple siRNA combination for reduction of target genes, collagen and alpha smooth muscle actin ( aSMA ) in rabbit corneas...targeting three key genes (TGFb, TGFbRII, and CTGF) that synergistically reduces the level of mRNAs for type I collagen gene and aSMA by >95% without

Entertainment-education and recruitment of cornea donors: the role of emotion and issue involvement.

Science.gov (United States)

Bae, Hyuhn-Suhck

2008-01-01

This study examined the role of emotional responses and viewer's level of issue involvement to an entertainment-education show about cornea donation in order to predict intention to register as cornea donors. Results confirmed that sympathy and empathy responses operated as a catalyst for issue involvement, which emerged as an important intermediary in the persuasion process. Issue involvement also was found to be a common causal antecedent of attitude, subjective norm, and perceived behavioral control, the last two of which predict intentions unlike attitude, which does not. The revised path model confirmed that involvement directly influences intention. The findings of this study suggest that adding emotion and involvement in the Theory of Planned Behavior (TPB) enhances the explanatory power of the theory in predicting intentions, which indicates the possibility of combining the Elaboration Likelihood Model (ELM) and the TPB in the prediction of human behaviors.

A porcine model of haematogenous brain infectionwith staphylococcus aureus

DEFF Research Database (Denmark)

Astrup, Lærke Boye; Agerholm, Jørgen Steen; Nielsen, Ole Lerberg

2012-01-01

A PORCINE MODEL OF HAEMATOGENOUS BRAIN INFECTION WITH STAPHYLOCOCCUS AUREUS Astrup Lærke1, Agerholm Jørgen1, Nielsen Ole1, Jensen Henrik1, Leifsson Páll1, Iburg Tine2. 1: Faculty of Health and Medical Sciences, University of Copenhagen, Denmark boye@life.ku.dk 2: National Veterinary Institute......, Uppsala, Sweden Introduction Staphylococcus aureus (S.aureus) is a common cause of sepsis and brain abscesses in man and a frequent cause of porcine pyaemia. Here we present a porcine model of haematogenous S. aureus-induced brain infection. Materials and Methods Four pigs had two intravenous catheters...... thromboemboli (two pigs). The venous catheter was used for blood sampling before, during and after inoculation. The pigs were euthanized either 24 or 48 hours after inoculation. The brains were collected and examined histologically. Results We describe unifocal suppurative encephalitis 48 hours after...

Molecular characterization of the porcine surfactant, pulmonary-associated protein C gene

DEFF Research Database (Denmark)

Cirera, S.; Nygård, A.B.; Jensen, H.E.

2006-01-01

The surfactant, pulmonary-associated protein C (SFTPC) is a peptide secreted by the alveolar type II pneumocytes of the lung. We have characterized the porcine SFTPC gene at genomic, transcriptional, and protein levels. The porcine SFTPC is a single-copy gene on pig chromosome 14. Two transcripts...

Secretion of pancreastatins from the porcine digestive tract

International Nuclear Information System (INIS)

Boerglum Jensen, T.D.; Holst, J.J.; Fahrenkrug, J.

1994-01-01

Pancreastatin, a 49-amino acid peptide with a COOH-terminal glycine amide, was originally isolated from porcine pancreas, but pancreastatin immunoreactivity has been found in several neuroendocrine tissues. There are strong indications that pancreastatin is derived from chromogranin A, since the amino acid sequence 240-288 in porcine chromogranin A corresponds to pancreastatin flanked by typical signals for proteolytic processing. The authors studied the effect of electric stimulation of the nervous supply to perfused porcine pancreas, antrum, nonantral stomach, and small intestine on the release of immunoreactive pancreastatin, and they have characterized the molecular nature of the secreted immunoreactivity by using a radioimmunoassay specific for the COOH-terminal glycine amide of porcine pancreastatin in combination with chromatography. In all tissues nerve stimulation significantly increased the release of immunoreactive pancreastatin. The secreted immunoreactive pancreastatin was heterogeneous, consisting of pancreastatin itself, a COOH-terminal pancreastatin fragment, and NH 2 -terminally extended pancreastatin forms. Pancreastatin predominated in the perfusate from pancreas and antrum, whereas mainly NH 2 -terminally extended molecular forms were secreted from the antrectomized stomach and small intestine. The different molecular forms of pancreastatin were secreted from the perfused organs in the same molar ratio as they occur in extracts of the corresponding tissues. Thus, pancreastatin and other chromogranin A-derived peptides in organ-specific proportions regularly accompany the secretion of the peptide hormones from the gastrointestinal tissues on appropriate stimulation. 40 refs., 5 figs

Desiccating Stress-Induced MMP Production and Activity Worsens Wound Healing in Alkali-Burned Corneas

Science.gov (United States)

Bian, Fang; Pelegrino, Flavia S. A.; Pflugfelder, Stephen C.; Volpe, Eugene A.; Li, De-Quan; de Paiva, Cintia S.

2015-01-01

Purpose To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury. Methods C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase–9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry. Results Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1β, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury. Conclusions Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation. PMID:26225631

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 3: Porcine islet product manufacturing and release testing criteria.

Science.gov (United States)

Rayat, Gina R; Gazda, Lawrence S; Hawthorne, Wayne J; Hering, Bernhard J; Hosking, Peter; Matsumoto, Shinichi; Rajotte, Ray V

2016-01-01

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the

A high-resolution comparative RH map of porcine chromosome (SSC) 2.

NARCIS (Netherlands)

Rattink, A.P.; Faivre, M.; Jungerius, B.J.; Groenen, M.A.M.; Harlizius, B.

2001-01-01

A high-resolution comparative map was constructed for porcine Chromosome (SSC) 2, where a QTL for back fat thickness (BFT) is located. A radiation hybrid (RH) map containing 33 genes and 25 microsatellite markers was constructed for this chromosome with a 3000-rad porcine RH panel. In total, 16

CT radiation dose and image quality optimization using a porcine model.

Science.gov (United States)

Zarb, Francis; McEntee, Mark F; Rainford, Louise

2013-01-01

To evaluate potential radiation dose savings and resultant image quality effects with regard to optimization of commonly performed computed tomography (CT) studies derived from imaging a porcine (pig) model. Imaging protocols for 4 clinical CT suites were developed based on the lowest milliamperage and kilovoltage, the highest pitch that could be set from current imaging protocol parameters, or both. This occurred before significant changes in noise, contrast, and spatial resolution were measured objectively on images produced from a quality assurance CT phantom. The current and derived phantom protocols were then applied to scan a porcine model for head, abdomen, and chest CT studies. Further optimized protocols were developed based on the same methodology as in the phantom study. The optimization achieved with respect to radiation dose and image quality was evaluated following data collection of radiation dose recordings and image quality review. Relative visual grading analysis of image quality criteria adapted from the European guidelines on radiology quality criteria for CT were used for studies completed with both the phantom-based or porcine-derived imaging protocols. In 5 out of 16 experimental combinations, the current clinical protocol was maintained. In 2 instances, the phantom protocol reduced radiation dose by 19% to 38%. In the remaining 9 instances, the optimization based on the porcine model further reduced radiation dose by 17% to 38%. The porcine model closely reflects anatomical structures in humans, allowing the grading of anatomical criteria as part of image quality review without radiation risks to human subjects. This study demonstrates that using a porcine model to evaluate CT optimization resulted in more radiation dose reduction than when imaging protocols were tested solely on quality assurance phantoms.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

Science.gov (United States)

Kim, Jung-Sik; Chung, Hyunwoo; Byun, Nari; Kang, Seong-Jun; Lee, Sunho; Shin, Jun-Seop; Park, Chung-Gyu

2018-03-04

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization. Copyright © 2018 Elsevier Inc. All rights reserved.

Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis

DEFF Research Database (Denmark)

Johansen, L K; Koch, J; Frees, D

2012-01-01

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), N...... dependent on the strain of bacteria inoculated and on the formation of a biofilm....... with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore...

T-style keratoprosthesis based on surface-modified poly (2-hydroxyethyl methacrylate) hydrogel for cornea repairs

International Nuclear Information System (INIS)

Xiang, Jun; Sun, Jianguo; Hong, Jiaxu; Wang, Wentao; Wei, Anji; Le, Qihua; Xu, Jianjiang

2015-01-01

Corneal disease is a common cause of blindness, and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents a novel T-style design of a keratoprosthesis and its preparation methods, in which a mechanically and structurally effective artificial cornea is made based on a poly(2-hydroxyethyl methacrylate) hydrogel. The porous skirt was modified with hyaluronic acid and cationized gelatin, and the bottom of the optical column was coated with poly(ethylene glycol). The physical properties of the T-style Kpro were analyzed using ultraviolet and visible spectrophotometry and electron scanning microscopy. The surface chemical properties were characterized using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The surface modification in the spongy skirt promoted cell adhesion and produced a firm bond between the corneal tissue and the implant device, while the surface modification in the optic column resisted cell adhesion and prevented retroprosthetic membrane formation. Through improved surgical techniques, the novel T-style keratoprosthesis provides enough mechanical stability to facilitate long-term biointegration with the host environment. In vivo implantation experiments showed that the T-style keratoprosthesis is a promising cornea alternative for patients with severe limbal stem cell deficiency and corneal opacity. - Highlights: • T-style keratoprosthesis was designed and prepared based on a PHEMA hydrogel. • Selective surface modifications effectively regulated cells' selective adhesion. • T-style keratoprosthesis provides enough mechanical stability to facilitate long-term biointegration with host tissues

T-style keratoprosthesis based on surface-modified poly (2-hydroxyethyl methacrylate) hydrogel for cornea repairs

Energy Technology Data Exchange (ETDEWEB)

Xiang, Jun [Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); Sun, Jianguo [Research Center, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); State Key Laboratory of Molecular Engineering of Polymers, Fudan University (China); Hong, Jiaxu [Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); Wang, Wentao [Research Center, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); Wei, Anji [Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); Le, Qihua [Research Center, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China); Xu, Jianjiang, E-mail: jianjiang-xu@163.com [Department of Ophthalmology, Eye & ENT Hospital, Shanghai Medical College, Fudan University (China); Key Laboratory of Myopia, Ministry of Health, Fudan University (China); Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University (China)

2015-05-01

Corneal disease is a common cause of blindness, and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents a novel T-style design of a keratoprosthesis and its preparation methods, in which a mechanically and structurally effective artificial cornea is made based on a poly(2-hydroxyethyl methacrylate) hydrogel. The porous skirt was modified with hyaluronic acid and cationized gelatin, and the bottom of the optical column was coated with poly(ethylene glycol). The physical properties of the T-style Kpro were analyzed using ultraviolet and visible spectrophotometry and electron scanning microscopy. The surface chemical properties were characterized using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The surface modification in the spongy skirt promoted cell adhesion and produced a firm bond between the corneal tissue and the implant device, while the surface modification in the optic column resisted cell adhesion and prevented retroprosthetic membrane formation. Through improved surgical techniques, the novel T-style keratoprosthesis provides enough mechanical stability to facilitate long-term biointegration with the host environment. In vivo implantation experiments showed that the T-style keratoprosthesis is a promising cornea alternative for patients with severe limbal stem cell deficiency and corneal opacity. - Highlights: • T-style keratoprosthesis was designed and prepared based on a PHEMA hydrogel. • Selective surface modifications effectively regulated cells' selective adhesion. • T-style keratoprosthesis provides enough mechanical stability to facilitate long-term biointegration with host tissues.

An Investigation of the Pathology and Pathogens Associated with Porcine Respiratory Disease Complex in Denmark

DEFF Research Database (Denmark)

Hansen, Mette Sif; Pors, S. E.; Jensen, H. E.

2010-01-01

), porcine reproductive and respiratory syndrome virus (both European and US type), porcine circovirus type 2 (PCV2), porcine respiratory coronavirus, porcine cytomegalovirus, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. All cases had cranioventral lobular bronchopneumonia consistent with PRDC....... There was a broad range of microscopical lesions and the cases were characterized as acute (n=10), subacute (n=24) or chronic (n=114) bronchopneumonia. Five bacterial species, five viruses and two Mycoplasma spp. were detected in different combinations. PCV2, M. hyopneumoniae, M. hyorhinis and Pasteurella multocida...

Antimicrobial compounds of porcine mucosa

Science.gov (United States)

Kotenkova, E. A.; Lukinova, E. A.; Fedulova, L. V.

2017-09-01

The aim of the study was to investigate porcine oral cavity mucosa (OCM), nasal cavity mucosa (NCM), rectal mucosa (RM) and tongue mucosa (TM) as sources of antimicrobial compounds. Ultrafiltrates with MW >30 kDa, MW 5-30 kDa and MW 30 kDa, the zone of microbial growth inhibition was 7.5 mm, for the MW<5 kDa fraction, it was 7 mm, and for MW 5-30 kDa fraction, it was 4.5 mm. No significant differences were found in high molecular weight proteomic profile, while qualitative and quantitative differences were observed in the medium and low molecular weight areas, especially in OCM and NCM. HPLC showed 221 tissue-specific peptides in OCM, 156 in NCM, 225 in RM, but only 5 in TM. The results observed confirmed porcine mucous tissues as a good source of antimicrobial compounds, which could be an actual alternative for reduction of microbial spoilage of foods.

Experimental study on the healing process following laser welding of the cornea.

Science.gov (United States)

Rossi, Francesca; Pini, Roberto; Menabuoni, Luca; Mencucci, Rita; Menchini, Ugo; Ambrosini, Stefano; Vannelli, Gabriella

2005-01-01

An experimental study evaluating the application of laser welding of the cornea and the subsequent healing process is presented. The welding of corneal wounds is achieved after staining the cut walls with a solution of the chromophore indocyanine green, and irradiating them with a diode laser (810 nm) operating at low power (60 to 90 mW). The result is a localized heating of the cut, inducing controlled welding of the stromal collagen. In order to optimize this technique and to study the healing process, experimental tests, simulating cataract surgery and penetrating keratoplasty, were performed on rabbits: conventional and laser-induced suturing of corneal wounds were thus compared. A follow-up study 7 to 90 days after surgery was carried out by means of objective and histological examinations, in order to optimize the welding technique and to investigate the subsequent healing process. The analyses of the laser-welded corneas evidenced a faster and more effective restoration of the architecture of the stroma. No thermal damage of the welded stroma was detected, nor were there foreign body reactions or other inflammatory processes. Copyright 2005 Society of Photo-Optical Instrumentation Engineers.

Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development

DEFF Research Database (Denmark)

Hall, Vanessa Jane; Christensen, Josef; Gao, Yu

2009-01-01

  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass. Copyright (c) 2009 Wiley-Liss, Inc.......  The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown. In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast. We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway...... (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4). We discovered two different expression profiles exist in the developing porcine embryo. The D6 porcine blastocyst (inner cell mass stage) is devoid...

Self-assembling colloidal system for the ocular administration of cyclosporine A.

Science.gov (United States)

Luschmann, Christoph; Tessmar, Joerg; Schoeberl, Simon; Strauß, Olaf; Luschmann, Karl; Goepferich, Achim

2014-01-01

In this study, we developed a self-assembling micellar system to deliver cyclosporine A (CsA) in an aqueous solution to the cornea. Two nonionic surfactants of the poly(ethylene glycol)-fatty alcohol ether type (Sympatens AS and Sympatens ACS) were characterized in terms of micelle size, shape, and charge, and their encapsulation efficiency for CsA. In an in situ single dose bioavailability study, the corneal CsA levels were determined in an enucleated porcine eye model. A commercial formulation and a 2% CsA olive oil solution served as references. Both surfactants formed spherical micelles with a size of 9 to 12 nm in water. A concentration as low as 0.3% (wt/vol) Sympatens AS was sufficient to entrap therapeutic levels of at least 0.1% (wt/vol) CsA. In the porcine in situ model, exceptionally high drug levels in the cornea were obtained for the micellar CsA solution (1557 ± 407 ngCsA/gcornea). They were significantly higher than those of Restasis (545 ± 137 ngCsA/gcornea) or the olive oil solution (452 ± 142 ngCsA/gcornea). In conclusion, we have shown a promising simple and efficient approach for the application of CsA in an aqueous solution to the cornea to treat inflammatory corneal diseases.

Molecular characterization, sequence analysis and tissue expression of a porcine gene – MOSPD2

Directory of Open Access Journals (Sweden)

Yang Jie

2017-01-01

Full Text Available The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2 of five species: horse (89%, human (90%, chimpanzee (89%, rhesus monkey (89% and mouse (85%; thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.

Concise classification of the genomic porcine endogenous retroviral gamma1 load to defined lineages.

Science.gov (United States)

Klymiuk, Nikolai; Wolf, Eckhard; Aigner, Bernhard

2008-02-05

We investigated the infection history of porcine endogenous retroviruses (PERV) gamma1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV gamma1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

The management of cornea blindness from severe corneal scarring, with the Athens Protocol (transepithelial topography-guided PRK therapeutic remodeling, combined with same-day, collagen cross-linking

Directory of Open Access Journals (Sweden)

Kanellopoulos AJ

2012-02-01

Full Text Available Anastasios John KanellopoulosLaservision.gr Institute, Athens, Greece; Manhattan Eye, Ear and Throat Hospital, New York, NY, USA; New York University Medical School, New York, NY, USAPurpose: To evaluate the safety and efficacy of combined transepithelial topography-guided photorefractive keratectomy (PRK therapeutic remodeling, combined with same-day, collagen cross-linking (CXL. This protocol was used for the management of cornea blindness due to severe corneal scarring.Methods: A 57-year-old man had severe corneal blindness in both eyes. Both corneas had significant central scars attributed to a firework explosion 45 years ago, when the patient was 12 years old. Corrected distance visual acuity (CDVA was 20/100 both eyes (OU with refraction: +4.00, –4.50 at 135° in the right eye and +3.50, –1.00 at 55° in the left. Respective keratometries were: 42.3, 60.4 at 17° and 35.8, 39.1 at 151.3°. Cornea transplantation was the recommendation by multiple cornea specialists as the treatment of choice. We decided prior to considering a transplant to employ the Athens Protocol (combined topography-guided partial PRK and CXL in the right eye in February 2010 and in the left eye in September 2010. The treatment plan for both eyes was designed on the topography-guided wavelight excimer laser platform.Results: Fifteen months after the right eye treatment, the right cornea had improved translucency and was topographically stable with uncorrected distance visual acuity (UDVA 20/50 and CDVA 20/40 with refraction +0.50, –2.00 at 5°. We noted a similar outcome after similar treatment applied in the left eye with UDVA 20/50 and CDVA 20/40 with –0.50, –2.00 at 170° at the 8-month follow-up.Conclusion: In this case, the introduction of successful management of severe cornea abnormalities and scarring with the Athens Protocol may provide an effective alternative to other existing surgical or medical options.Keywords: Athens Protocol, collagen cross

Temperature profiles of different cooling methods in porcine pancreas procurement.

Science.gov (United States)

Weegman, Bradley P; Suszynski, Thomas M; Scott, William E; Ferrer Fábrega, Joana; Avgoustiniatos, Efstathios S; Anazawa, Takayuki; O'Brien, Timothy D; Rizzari, Michael D; Karatzas, Theodore; Jie, Tun; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2014-01-01

Porcine islet xenotransplantation is a promising alternative to human islet allotransplantation. Porcine pancreas cooling needs to be optimized to reduce the warm ischemia time (WIT) following donation after cardiac death, which is associated with poorer islet isolation outcomes. This study examines the effect of four different cooling Methods on core porcine pancreas temperature (n = 24) and histopathology (n = 16). All Methods involved surface cooling with crushed ice and chilled irrigation. Method A, which is the standard for porcine pancreas procurement, used only surface cooling. Method B involved an intravascular flush with cold solution through the pancreas arterial system. Method C involved an intraductal infusion with cold solution through the major pancreatic duct, and Method D combined all three cooling Methods. Surface cooling alone (Method A) gradually decreased core pancreas temperature to <10 °C after 30 min. Using an intravascular flush (Method B) improved cooling during the entire duration of procurement, but incorporating an intraductal infusion (Method C) rapidly reduced core temperature 15-20 °C within the first 2 min of cooling. Combining all methods (Method D) was the most effective at rapidly reducing temperature and providing sustained cooling throughout the duration of procurement, although the recorded WIT was not different between Methods (P = 0.36). Histological scores were different between the cooling Methods (P = 0.02) and the worst with Method A. There were differences in histological scores between Methods A and C (P = 0.02) and Methods A and D (P = 0.02), but not between Methods C and D (P = 0.95), which may highlight the importance of early cooling using an intraductal infusion. In conclusion, surface cooling alone cannot rapidly cool large (porcine or human) pancreata. Additional cooling with an intravascular flush and intraductal infusion results in improved core porcine pancreas temperature profiles during procurement and

A sensitive duplex nanoparticle-assisted PCR assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens.

Science.gov (United States)

Zhu, Yu; Liang, Lin; Luo, Yakun; Wang, Guihua; Wang, Chunren; Cui, Yudong; Ai, Xia; Cui, Shangjin

2017-02-01

In this study, a novel duplex nanoparticle-assisted polymerase chain reaction (nanoPCR) assay was developed to detect porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV). Two pairs of primers were designed based on the conserved region within the N gene of PEDV and TGEV. In a screening of 114 clinical samples from four provinces in China for PEDV and TGEV, 48.2 and 3.5 % of the samples, respectively, tested positive. Under optimized conditions, the duplex nanoPCR assay had a detection limit of 7.6 × 10 1 and 8.5 × 10 1 copies μL -1 for PEDV and TGEV, respectively. The sensitivity of the duplex nanoPCR assay was ten times higher than that of a conventional PCR assay. Moreover, no fragments were amplified when the duplex nanoPCR assay was used to test samples containing other porcine viruses. Our results indicate that the duplex nanoPCR assay described here is useful for the rapid detection of PEDV and TGEV and can be applied in clinical diagnosis.

Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.

Directory of Open Access Journals (Sweden)

Hassan Alizadeh

Full Text Available Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK, a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05 CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

Tissue Sampling Guides for Porcine Biomedical Models.

Science.gov (United States)

Albl, Barbara; Haesner, Serena; Braun-Reichhart, Christina; Streckel, Elisabeth; Renner, Simone; Seeliger, Frank; Wolf, Eckhard; Wanke, Rüdiger; Blutke, Andreas

2016-04-01

This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from ∼50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology; immunohistochemistry;in situhybridization; electron microscopy; and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results. © The Author(s) 2016.

Purification, characterization and immunolocalization of porcine surfactant protein D

DEFF Research Database (Denmark)

Sørensen, C.M.; Nielsen, Ove Lilholm; Willis, A.

2005-01-01

in a dose and Ca2+-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting......Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability to bind complex...... glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity...

Identification of the porcine homologous of human disease causing trinucleotide repeat sequences

DEFF Research Database (Denmark)

Madsen, Lone Bruhn; Thomsen, Bo; Sølvsten, Christina Ane Elisabeth

2007-01-01

in this paper the identification of porcine noncoding and polyglutamine-encoding TNR regions and the comparison to the homologous TNRs from human, chimpanzee, dog, opossum, rat, and mouse. Several of the porcine TNR regions are highly polymorphic both within and between different breeds. The TNR regions...

Effect of irradiation and cystamine on the secondary induction of cornea in anura larvae

International Nuclear Information System (INIS)

Popov, D.V.; Starodubov, S.M.

1977-01-01

Secondary induction of cornea in the transplanted skin has been studied in amphibian larvae after the effect of irradiation and cystamine on the recipients and donors. Comparison of the results obtained with those reported earlier has demonstrated that the irradiated tissue is more responsive to inductive effects than the intact one

Biofunctionalized Lysophosphatidic Acid/Silk Fibroin Film for Cornea Endothelial Cell Regeneration

Science.gov (United States)

Jeon, Hayan; Oliveira, Joaquim Miguel; Reis, Rui Luis; Khang, Gilson

2018-01-01

Cornea endothelial cells (CEnCs) tissue engineering is a great challenge to repair diseased or damaged CEnCs and require an appropriate biomaterial to support cell proliferation and differentiation. Biomaterials for CEnCs tissue engineering require biocompatibility, tunable biodegradability, transparency, and suitable mechanical properties. Silk fibroin-based film (SF) is known to meet these factors, but construction of functionalized graft for bioengineering of cornea is still a challenge. Herein, lysophosphatidic acid (LPA) is used to maintain and increase the specific function of CEnCs. The LPA and SF composite film (LPA/SF) was fabricated in this study. Mechanical properties and in vitro studies were performed using a rabbit model to demonstrate the characters of LPA/SF. ATR-FTIR was characterized to identify chemical composition of the films. The morphological and physical properties were performed by SEM, AFM, transparency, and contact angle. Initial cell density and MTT were performed for adhesion and cell viability in the SF and LPA/SF film. Reverse transcription polymerase chain reactions (RT-PCR) and immunofluorescence were performed to examine gene and protein expression. The results showed that films were designed appropriately for CEnCs delivery. Compared to pristine SF, LPA/SF showed higher biocompatibility, cell viability, and expression of CEnCs specific genes and proteins. These indicate that LPA/SF, a new biomaterial, offers potential benefits for CEnCs tissue engineering for regeneration. PMID:29710848

Molecular Hydrogen Effectively Heals Alkali-Injured Cornea via Suppression of Oxidative Stress

Czech Academy of Sciences Publication Activity Database

Čejka, Čestmír; Kössl, Jan; Heřmánková, Barbora; Holáň, Vladimír; Čejková, Jitka

2017-01-01

Roč. 2017, č. 2017 (2017), s. 8906027 ISSN 1942-0900 R&D Projects: GA ČR(CZ) GA14-12580S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1508; GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : 4-coumaric acid * rabbit cornea * nitric-oxide Subject RIV: FF - HEENT, Dentistry OBOR OECD: Ophthalmology Impact factor: 4.593, year: 2016

An anatomical study of porcine peripheral nerve and its potential use in nerve tissue engineering

Science.gov (United States)

Zilic, Leyla; Garner, Philippa E; Yu, Tong; Roman, Sabiniano; Haycock, John W; Wilshaw, Stacy-Paul

2015-01-01

Current nerve tissue engineering applications are adopting xenogeneic nerve tissue as potential nerve grafts to help aid nerve regeneration. However, there is little literature that describes the exact location, anatomy and physiology of these nerves to highlight their potential as a donor graft. The aim of this study was to identify and characterise the structural and extracellular matrix (ECM) components of porcine peripheral nerves in the hind leg. Methods included the dissection of porcine nerves, localisation, characterisation and quantification of the ECM components and identification of nerve cells. Results showed a noticeable variance between porcine and rat nerve (a commonly studied species) in terms of fascicle number. The study also revealed that when porcine peripheral nerves branch, a decrease in fascicle number and size was evident. Porcine ECM and nerve fascicles were found to be predominately comprised of collagen together with glycosaminoglycans, laminin and fibronectin. Immunolabelling for nerve growth factor receptor p75 also revealed the localisation of Schwann cells around and inside the fascicles. In conclusion, it is shown that porcine peripheral nerves possess a microstructure similar to that found in rat, and is not dissimilar to human. This finding could extend to the suggestion that due to the similarities in anatomy to human nerve, porcine nerves may have utility as a nerve graft providing guidance and support to regenerating axons. PMID:26200940

Porcine cluster of differentiation (CD) markers 2018 update.

Science.gov (United States)

Dawson, Harry D; Lunney, Joan K

2018-06-01

Pigs are a major source of food worldwide; preventing and treating their infectious diseases is essential, requiring a thorough understanding of porcine immunity. The use of pigs as models for human physiology is a growing area; progress in this area has been limited because the immune toolkit is not robust. The international community has established cluster of differentiation (CD) markers for assessing cells involved in immunity as well as characterizing numerous other cells like stem cells. Overall, for humans 419 proteins have been designated as CD markers, each reacting with a defined set of antibodies (Abs). This paper summarizes current knowledge of swine CD markers and identifies 359 corresponding CD proteins in pigs. A broad-based literature and vendor search was conducted to identify defined sets of monoclonal (mAbs) and polyclonal Abs (pAbs) reacting with porcine CD markers along with other reagents (fusion proteins, ELISAs, PCR assays, and gene edited cell and pig models). This process identified over 800 reagents that are reportedly reactive with 266 pig CD markers. Despite this number, there is a great need to develop and characterize additional CD marker reagents, particularly mAbs, for pig research. There are numerous high priority targets: reagents for the characterization of porcine innate lymphoid cells, polarized macrophages and T regulatory cells and for the detection of porcine CD45 isoforms. Overall, improved technologies and genomics have contributed to dramatic increases in our knowledge of the pig, its immune system, disease and vaccine responses, and utility as a biomedical model. The development of more CD reagents will clearly advance these initiatives. Published by Elsevier Ltd.

Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

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Hause, Ben M; Collin, Emily A; Peddireddi, Lalitha; Yuan, Fangfeng; Chen, Zhenhai; Hesse, Richard A; Gauger, Phillip C; Clement, Travis; Fang, Ying; Anderson, Gary

2015-10-01

Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

Porcine, murine and human sialoadhesin (Sn/Siglec-1/CD169): portals for porcine reproductive and respiratory syndrome virus entry into target cells.

Science.gov (United States)

Van Breedam, Wander; Verbeeck, Mieke; Christiaens, Isaura; Van Gorp, Hanne; Nauwynck, Hans J

2013-09-01

Porcine sialoadhesin (pSn; a sialic acid-binding lectin) and porcine CD163 (pCD163) are molecules that facilitate infectious entry of porcine reproductive and respiratory syndrome virus (PRRSV) into alveolar macrophages. In this study, it was shown that murine Sn (mSn) and human Sn (hSn), like pSn, can promote PRRSV infection of pCD163-expressing cells. Intact sialic acid-binding domains are crucial, since non-sialic acid-binding mutants of pSn, mSn and hSn did not promote infection. Endodomain-deletion mutants of pSn, mSn and hSn promoted PRRSV infection less efficiently, but also showed markedly reduced expression levels, making further research into the potential role of the Sn endodomain in PRRSV receptor activity necessary. These data further complement our knowledge on Sn as an important PRRSV receptor, and suggest - in combination with other published data - that species differences in the main PRRSV entry mediators Sn and CD163 do not account for the strict host species specificity displayed by the virus.

Enteric porcine viruses in farmed shellfish in Denmark.

Science.gov (United States)

Krog, J S; Larsen, L E; Schultz, A C

2014-09-01

Bivalve shellfish are at constant risk of being exposed to pathogens as a consequence of contamination of the shellfish beds with human or animal waste originating from sewage treatment plants or slurry fertilized fields. Consumption of contaminated oysters and mussels are frequently reported as causes of disease outbreaks caused by norovirus or hepatitis A virus. Other zoonotic pathogens such as hepatitis E virus (HEV), rotavirus (RV) and Salmonella from livestock may also be transmitted to shellfish via this route. In this study, 29 pooled samples from commercial Danish blue mussels were tested for porcine pathogens and indicator bacteria Escherichia coli (E. coli). All samples tested negative for HEV, RV and Salmonella, whereas E. coli and the highly stable porcine circovirus type 2 (PCV2) were detected in eight and 12 samples, respectively. This is the first study to report the detection of PCV2 in commercial mussels. Based on the detection of PCV2 in clean areas with low prevalence of the normally applied fecal indicator E. coli, testing for PCV2 may be a more sensitive and robust specific porcine waste indicator in shellfish harvesting areas. Copyright © 2014. Published by Elsevier B.V.

The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.

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Hermel, Martin; Salla, Sabine; Fuest, Matthias; Walter, Peter

2017-03-01

Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. I-ECD was 2812 ± 360/mm 2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm 2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm 2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve

The Resin-Embedded Cornea Prepared Via Rapid Processing Protocol : A Good Histomorphometric Target for Clinical Investigation in Ophthalmology and Optometry

Science.gov (United States)

Cheah, Pike See; Mohidin, Norhani; Mohd Ali, Bariah; Maung, Myint; Latif, Azian Abdul

2008-01-01

This study illustrates and quantifies the changes on corneal tissue between the paraffin-embedded and resin-embedded blocks and thus, selects a better target in investigational ophthalmology and optometry via light microscopy. Corneas of two cynomolgus monkeys (Macaca fascicularis) were used in this study. The formalin-fixed cornea was prepared in paraffin block via the conventional tissue processing protocol (4-day protocol) and stained with haematoxylin and eosin. The glutaraldehyde-fixed cornea was prepared in resin block via the rapid and modified tissue processing procedure (1.2-day protocol) and stained with toluidine blue. The paraffin-embedded sample exhibits various undesired tissue damage and artifact such as thinner epithelium (due to the substantial volumic extraction from the tissue), thicker stroma layer (due to the separation of lamellae and the presence of voids) and the distorted endothelium. In contrast, the resin-embedded corneal tissue has demonstrated satisfactory corneal ultrastructural preservation. The rapid and modified tissue processing method for preparing the resin-embedded is particularly beneficial to accelerate the microscopic evaluation in ophthalmology and optometry. PMID:22570589

The comparative safety of genipin versus UVA-riboflavin crosslinking of rabbit corneas

Science.gov (United States)

Song, Wenjing; Tang, Yun; Qiao, Jing; Li, Haili; Rong, Bei; Yang, Songlin; Wu, Yuan

2017-01-01

Purpose To investigate, after 24 h, the safety of genipin or ultraviolet A (UVA)-riboflavin crosslinking of keratocytes and endothelial cells. Methods Fifteen New Zealand white rabbits were selected and divided into a PBS group (five rabbits), a 0.2% genipin crosslinking (GP-CXL) group (five rabbits), and a UVA-riboflavin crosslinking (UVA-CXL) group (five rabbits). In the GP-CXL and PBS groups, 0.2% genipin or PBS was applied to the corneal surface of the right eyes. In the UVA-CXL group, a clinical crosslinking procedure was used. Before and after surgery, the operated eyes of each group were characterized with confocal microscopy, and the corneal buttons were excised for endothelium staining and electron microscopy. Results The corneal endothelial cell density of the GP-CXL, UVA-CLX, and PBS groups changed. There was a statistically significant difference in thickness and changes in corneal endothelial cell density between the UVA-CXL group and the PBS group (pUVA-CXL group and the GP-CXL group (pUVA-CXL group. In the GP-CXL group, only active keratocytes were found and minimal endothelial cell damage. Conclusions Treatment of rabbit corneas with 0.2% genipin showed minimal toxicity toward keratocytes and endothelial cells. Genipin is safer than UVA-CXL for crosslinking of thin corneas. PMID:28761323

Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

Science.gov (United States)

Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

2015-03-01

A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway

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Wei Zhang

2018-02-01

Full Text Available Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs, are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX. These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

Collagen cross-linking in thin corneas

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Prema Padmanabhan

2013-01-01

Full Text Available Collagen cross-linking (CXL has become the standard of care for progressive keratoconus, after numerous clinical studies have established its efficacy and safety in suitably selected eyes. The standard protocol is applicable in eyes which have a minimum corneal thickness of 400 μm after epithelial debridement. This prerequisite was stipulated to protect the corneal endothelium and intraocular tissues from the deleterious effect of ultraviolet-A (UVA radiation. However, patients with keratoconus often present with corneal thickness of less than 400 μm and could have otherwise benefited from this procedure. A few modifications of the standard procedure have been suggested to benefit these patients without a compromise in safety. Transepithelial cross-linking, pachymetry-guided epithelial debridement before cross-linking, and the use of hypoosmolar riboflavin are some of the techniques that have been attempted. Although clinical data is limited at the present time, these techniques are worth considering in patients with thin corneas. Further studies are needed to scientifically establish their efficacy and safety.

EFFECT OF NATURAL PLANT EXTRACTS ON PORCINE OVARIAN FUNCTIONS

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Attila Kádasi

2015-02-01

Full Text Available This report provides information about the impact of chosen natural plant extracts on basic ovarian functions. This article summarizes our results concerning the effect of selected plant extracts on proliferation, apoptosis and hormone secretion – release of progesterone (P4, testosterone (T and leptin (L on porcine granulosa cells (GC, We analyzed effects of ginkgo (GB, rooibos (RB, flaxseed (FL, green tea polyphenols (GTPP, green tea - epigallocatechin-3-gallate (EGCG, resveratrol (RSV and curcumin (CURC (0; 1; 10 and 100 μg.ml-1 on markers of proliferation, apoptosis and secretory activity of porcine ovarian granulosa cells by using immunocytochemistry and EIA. It was demonstrated, that all these natural plants and plant molecules inhibited the accumulation of proliferation-related peptide (PCNA and apoptosis-associated peptide (Bax in cultured. Furthermore, it was observed that natural plant extracts altered progesterone, testosterone and leptin release in porcine ovarian cells. It is concluded, that GB, RB, FL, RSV, CURC, GTPP and EGCG can directly affect ovarian cells and therefore they could potentially influence ovarian functions.

Proliferative activity of endotheliocytes of growing capillaries of the rabbit cornea

International Nuclear Information System (INIS)

Gurina, O.Yu.; Mamontov, S.G.; Banin, V.V.

1987-01-01

The authors studied the intensity of DNA synthesis by cells of newly formed capillaries, growing in the rabbit cornea, after infliction of a silver nitrate burn and local application of colchicine. The intensity of capillary growth was investigated during stimulation and a combination of the burn with colchicine. Changes in activity of DNA synthesis by the endotheliocytes of newly formed capillaries during exposure throughout growth were also investigated. The intensity of cell proliferation was studied by measuring the incorporation of tritium-labelled thymidine into the endotheoiocyte nuclei

Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

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Brendan Mulligan

2012-12-01

Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

Molecular characterization and analysis of the porcine NURR1 gene

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Knud Larsen

2016-12-01

Here we report the isolation and characterization of porcine NURR1 cDNA. The NURR1 cDNA was RT-PCR cloned using NURR1-specific oligonucleotide primers derived from in silico sequences. The porcine NURR1 cDNA encodes a polypeptide of 598 amino acids, displaying a very high similarity with bovine, human and mouse (99% NURR1 protein. Expression analysis revealed a differential NURR1 mRNA expression in various organs and tissues. NURR1 transcripts could be detected as early as at 60 days of embryo development in different brain tissues. A significant increase in NURR1 transcript in the cerebellum and a decrease in NURR1 transcript in the basal ganglia was observed during embryo development. The porcine NURR1 gene was mapped to chromosome 15. Two missense mutations were found in exon 3, the first coding exon of NURR1. Methylation analysis of the porcine NURR1 gene body revealed a high methylation degree in brain tissue, whereas methylation of the promoter was very low. A decrease in DNA methylation in a discrete region of the NURR1 promoter was observed in pig frontal cortex during pig embryo development. This observation correlated with an increase in NURR1 transcripts. Therefore, methylation might be a determinant of NURR1 expression at certain time points in embryo development.

Tissue Engineering the Cornea: The Evolution of RAFT

Science.gov (United States)

Levis, Hannah J.; Kureshi, Alvena K.; Massie, Isobel; Morgan, Louise; Vernon, Amanda J.; Daniels, Julie T.

2015-01-01

Corneal blindness affects over 10 million people worldwide and current treatment strategies often involve replacement of the defective layer with healthy tissue. Due to a worldwide donor cornea shortage and the absence of suitable biological scaffolds, recent research has focused on the development of tissue engineering techniques to create alternative therapies. This review will detail how we have refined the simple engineering technique of plastic compression of collagen to a process we now call Real Architecture for 3D Tissues (RAFT). The RAFT production process has been standardised, and steps have been taken to consider Good Manufacturing Practice compliance. The evolution of this process has allowed us to create biomimetic epithelial and endothelial tissue equivalents suitable for transplantation and ideal for studying cell-cell interactions in vitro. PMID:25809689

The management of cornea blindness from severe corneal scarring, with the Athens Protocol (transepithelial topography-guided PRK therapeutic remodeling, combined with same-day, collagen cross-linking).

Science.gov (United States)

Kanellopoulos, Anastasios John

2012-01-01

To evaluate the safety and efficacy of combined transepithelial topography-guided photorefractive keratectomy (PRK) therapeutic remodeling, combined with same-day, collagen cross-linking (CXL). This protocol was used for the management of cornea blindness due to severe corneal scarring. A 57-year-old man had severe corneal blindness in both eyes. Both corneas had significant central scars attributed to a firework explosion 45 years ago, when the patient was 12 years old. Corrected distance visual acuity (CDVA) was 20/100 both eyes (OU) with refraction: +4.00, -4.50 at 135° in the right eye and +3.50, -1.00 at 55° in the left. Respective keratometries were: 42.3, 60.4 at 17° and 35.8, 39.1 at 151.3°. Cornea transplantation was the recommendation by multiple cornea specialists as the treatment of choice. We decided prior to considering a transplant to employ the Athens Protocol (combined topography-guided partial PRK and CXL) in the right eye in February 2010 and in the left eye in September 2010. The treatment plan for both eyes was designed on the topography-guided wavelight excimer laser platform. Fifteen months after the right eye treatment, the right cornea had improved translucency and was topographically stable with uncorrected distance visual acuity (UDVA) 20/50 and CDVA 20/40 with refraction +0.50, -2.00 at 5°. We noted a similar outcome after similar treatment applied in the left eye with UDVA 20/50 and CDVA 20/40 with -0.50, -2.00 at 170° at the 8-month follow-up. In this case, the introduction of successful management of severe cornea abnormalities and scarring with the Athens Protocol may provide an effective alternative to other existing surgical or medical options.

Significant Down-Regulation of “Biological Adhesion” Genes in Porcine Oocytes after IVM

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Joanna Budna

2017-12-01

Full Text Available Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes’ maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR (“before IVM” group, or first in vitro matured and then if classified as BCB+ passed to molecular analyses (“after IVM” group. As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes’ successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte’s achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.

Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

Science.gov (United States)

Sun, Huanxing; Zhu, Yangyang; Pan, Hongyi; Chen, Xiaosong; Balestrini, Jenna L; Lam, TuKiet T; Kanyo, Jean E; Eichmann, Anne; Gulati, Mridu; Fares, Wassim H; Bai, Hanwen; Feghali-Bostwick, Carol A; Gan, Ye; Peng, Xueyan; Moore, Meagan W; White, Eric S; Sava, Parid; Gonzalez, Anjelica L; Cheng, Yuwei; Niklason, Laura E; Herzog, Erica L

2016-05-01

Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were

Appraisal of the porcine kidney autotransplantation model

NARCIS (Netherlands)

Post, Ivo C. J. H.; Dirkes, Marcel C.; Heger, Michal; van Loon, Johannes P. A. M.; Swildens, Bas; Huijzer, Goos M.; van Gulik, Thomas M.

2012-01-01

Animal models are extensively used for transplantation related research, especially kidney transplantation. Porcine autotransplantation models are considered to be favorable regarding translatability to the human setting. The key determinants for translatability of the model are discussed,

Moorfields technique of donor cornea mounting for femtosecond-assisted keratoplasty: use of viscoelastic in the artificial anterior chamber.

Science.gov (United States)

Iovieno, Alfonso; Chowdhury, Vivek; Stevens, Julian D; Maurino, Vincenzo

2012-07-01

Appropriate mounting and cutting of the donor sclero-corneal cap is often cumbersome during femtosecond laser-assisted keratoplasty. The authors describe a technique for donor cornea femtosecond laser cutting using ophthalmic viscoelastic devices. The donor sclero-corneal cap is mounted on the artificial anterior chamber using a dispersive ophthalmic viscoelastic device instead of saline solution. The chances of artificial anterior chamber pressure loss, inadequate applanation, and fluid leaks are consistently reduced with the use of dispersive ophthalmic viscoelastic devices. The speed of donor femtosecond laser cutting is increased. The viscosity and elasticity of dispersive ophthalmic viscoelastic devices greatly assist the procedure with regard to ease of applanation, corneal endothelium protection, and decreased distortion of the applanated cornea. Copyright 2012, SLACK Incorporated.

Comparison of the effect of fatty alcohols on the permeation of melatonin between porcine and human skin.

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Andega, S; Kanikkannan, N; Singh, M

2001-11-09

Melatonin (MT) is a hormone secreted by the pineal gland that plays an important role in the regulation of the circadian sleep-wake cycle. It would be advantageous to administer MT using a transdermal delivery system for the treatment of sleep disorders such as delayed sleep syndrome, jet lag in travelers, cosmonauts and shift workers. The porcine skin has been found to have similar morphological and functional characteristics as human skin. The elastic fibres in the dermis, enzyme pattern of the epidermis, epidermal tissue turnover time, keratinous proteins and thickness of epidermis of porcine skin are similar to human skin. However, the fat deposition and vascularisation of the cutaneous glands of porcine skin are different from human skin. In addition, porcine skin has been found to have a close permeability character to human skin. However, the comparative effect of chemical penetration enhancers on the permeation of drugs between porcine and human skin has not been reported. The purpose of this study was to compare the effect of fatty alcohols on the permeability of porcine and human skin using MT as a model compound. The effect of saturated fatty alcohols (octanol, nonanol, decanol, undecanol, lauryl alcohol, tridecanol, myristyl alcohol) and unsaturated fatty alcohols (oleyl alcohol, linoleyl alcohol, linolenyl alcohol) at 5% concentration was tested across dermatomed porcine and human skin. Our studies showed a parabolic relationship between the carbon chain length of saturated fatty alcohols and permeation enhancement of MT with both porcine and human skin. Maximum permeation of MT was observed when fatty alcohol carbon chain length was 10. In general, as the level of unsaturation increased from one to two double bonds, there was an increase in the permeation of MT both in porcine and human skin. However, a decrease in the permeation was observed with three double bonds. Regression analysis using the steady state flux data showed a significant positive

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas

Directory of Open Access Journals (Sweden)

Belquiz Amaral Nassaralla

2013-06-01

Full Text Available PURPOSE: To evaluate the thinnest corneal thickness changes during and after corneal collagen cross-linking treatment with ultraviolet-A irradiation, using hypo-osmolar riboflavin solution in thin corneas. METHODS: Eighteen eyes of 18 patients were included in this study. After epithelium removal, iso-osmolar 0.1% riboflavin solution was instilled to the cornea every 3 minutes for 30 minutes. Hypo-osmolar 0.1% riboflavin solution was then applied every 20 seconds for 5 minutes or until the thinnest corneal thickness reached 400 µm. Ultraviolet-A irradiation was performed for 30 minutes. During irradiation, iso-osmolar 0.1% riboflavin drops were applied every 5 minutes. Ultrasound pachymetry was performed at approximately the thinnest point of the cornea preoperatively, after epithelial removal, after iso-osmolar riboflavin instillation, after hypo-osmolar riboflavin instillation, after ultraviolet-A irradiation, and at 1, 6 and 12 months after treatment. RESULTS: Mean preoperative thinnest corneal thickness was 380 ± 11 µm. After epithelial removal it decreased to 341 ± 11 µm, and after 30 minutes of iso-osmolar 0.1% riboflavin drops, to 330 ± 7.6 µm. After hypo-osmolar 0.1% riboflavin drops, mean thinnest corneal thickness increased to 418 ± 11 µm. After UVA irradiation, it was 384 ± 10 µm. At 1, 6 and 12 months after treatment, it was 372 ± 10 µm, 381 ± 12.7, and 379 ± 15 µm, respectively. No intraoperative, early postoperative, or late postoperative complications were noted. CONCLUSIONS: Hypo-osmolar 0.1% riboflavin solution seems to be effective for swelling thin corneas. The swelling effect is transient and short acting. Corneal thickness should be monitored throughout the procedure. Larger sample sizes and longer follow-up are required in order to make meaningful conclusions regarding safety.

New Technologies for Surgery of the Congenital Cardiac Defect

Directory of Open Access Journals (Sweden)

David Kalfa

2013-07-01

Full Text Available The surgical repair of complex congenital heart defects frequently requires additional tissue in various forms, such as patches, conduits, and valves. These devices often require replacement over a patient’s lifetime because of degeneration, calcification, or lack of growth. The main new technologies in congenital cardiac surgery aim at, on the one hand, avoiding such reoperations and, on the other hand, improving long-term outcomes of devices used to repair or replace diseased structural malformations. These technologies are: 1 new patches: CorMatrix® patches made of decellularized porcine small intestinal submucosa extracellular matrix; 2 new devices: the Melody® valve (for percutaneous pulmonary valve implantation and tissue-engineered valved conduits (either decellularized scaffolds or polymeric scaffolds; and 3 new emerging fields, such as antenatal corrective cardiac surgery or robotically assisted congenital cardiac surgical procedures. These new technologies for structural malformation surgery are still in their infancy but certainly present great promise for the future. But the translation of these emerging technologies to routine health care and public health policy will also largely depend on economic considerations, value judgments, and political factors.

Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

DEFF Research Database (Denmark)

Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

2013-01-01

from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

Directory of Open Access Journals (Sweden)

Li Bin

Full Text Available Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

Science.gov (United States)

Bin, Li; Luping, Du; Bing, Sun; Zhengyu, Yu; Maojun, Liu; Zhixin, Feng; Yanna, Wei; Haiyan, Wang; Guoqing, Shao; Kongwang, He

2014-01-01

Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod)-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO_2) thin films

International Nuclear Information System (INIS)

Nordin, N.; Azizah, N.; Hashim, U.

2016-01-01

Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO_2) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO2) thin films

Science.gov (United States)

Nordin, N.; Hashim, U.; Azizah, N.

2016-07-01

Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO2) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

Science.gov (United States)

Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

2018-06-01

Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The effect of radiation and cystamine on the ability of the eye to induce the cornea in the intact skin

International Nuclear Information System (INIS)

Popov, D.V.; Starodubov, S.M.

1975-01-01

Common frog tadpoles (with and without cystamine protection) were subjected to whole-body irradiation (700 R) and skin transplanted from the back of donors to the site of the outer cornea of the right eye of the recipients. Secondary cornea induction in non-irradiated skin transplanted to the eye was then studied. It was found that a dose of 700 R completely suppresses the induction capacity of the eye, while cystamine protects it to 47.8%. It was also shown that the irradiation of tadpoles with X-rays slows down their general development, but that, with cystamine protection, their development rate remains normal. (V.A.P.)

Xanthelasma Palpebrarum with Arcus Cornea: A Clinical and Biochemical Study.

Science.gov (United States)

Nair, Pragya Ashok; Patel, Chaitali R; Ganjiwale, Jaishree D; Diwan, Nilofar Gulamsha; Jivani, Nidhi Bhimjibhai

2016-01-01

Xanthelasma palpebrarum (XP) is characterized by sharply demarcated yellowish flat plaques on upper and lower eyelids. It is commonly seen in women with a peak incidence at 30-50 years. It is also considered as the cutaneous marker of underlying atherosclerosis along with the disturbed lipid metabolism. XP and corneal arcus are associated with increased levels of serum cholesterol and low-density lipoprotein (LDL) cholesterol. To study the clinical pattern of XP, its relationship with lipid profile and association with arcus cornea. This study was conducted at Department of Dermatology and Opthalmology, between August 2013 and January 2015. Patients with clinical diagnosis of XP who visited skin outpatient department and willing to undergo lipid profile test and eye examination were included in the study. Data regarding demographics, clinical findings, family history, and past history were noted along with the lipid profile details. Data of age-matched healthy controls were taken for comparison. The clinical profile of the participants was presented using frequency and proportions. Gender wise analysis comparing the lipid profile in cases with XP and without XP was done using independent sample t-test. Total 49 patients of XP, 81.6% were females. Maximum, 35% patients were among 50-60 years of age and 69.4% were homemakers by occupation. The average lipid values were-cholesterol 210.57 mg%, triglyceride 123.06 mg%. LDL 142.79 mg% and VLDL 30.95 mg% among patients of XP. Arcus cornea was found in 20% cases of XP. Patients of XP requires proper investigation at the onset and regular follow-up thereafter for any altered lipid profile and early diagnosis of coronary artery disease.

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

Science.gov (United States)

Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

2016-12-12

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

Characterisation of the porcine eyeball as an in-vitro model for dry eye.

Science.gov (United States)

Menduni, Francesco; Davies, Leon N; Madrid-Costa, D; Fratini, Antonio; Wolffsohn, James S

2018-02-01

To characterise the anatomical parameters of the porcine eye for potentially using it as a laboratory model of dry eye. Anterior chamber depth and angle, corneal curvature, shortest and longest diameter, endothelial cell density, and pachymetry were measured in sixty freshly enucleated porcine eyeballs. Corneal steepest meridian was 7.85±0.32mm, corneal flattest meridian was 8.28±0.32mm, shortest corneal diameter was 12.69±0.58mm, longest corneal diameter was 14.88±0.66mm and central corneal ultrasonic pachymetry was 1009±1μm. Anterior chamber angle was 28.83±4.16°, anterior chamber depth was 1.77±0.27mm, and central corneal thickness measured using OCT was 1248±144μm. Corneal endothelial cell density was 3250±172 cells/mm 2 . Combining different clinical techniques produced a pool of reproducible data on the porcine eye anatomy, which can be used by researchers to assess the viability of using the porcine eye as an in-vitro/ex-vivo model for dry eye. Due to the similar morphology with the human eye, porcine eyeballs may represent a useful and cost effective model to individually study important key factors in the development of dry eye, such as environmental and mechanical stresses. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

International Nuclear Information System (INIS)

Lenich, A.C.

1985-01-01

Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5- 14 C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively

Cloning and expression of porcine SRPK1 gene

African Journals Online (AJOL)

Academic Journals

2012-01-10

Jan 10, 2012 ... different porcine tissue and skeletal muscle repair processes. ... Biology Engineering Technology Service Co., Ltd; while ethanol, agarose gel DNA ..... muscle fiber regeneration after bupivacaine hydrochloride-and acid.

Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

International Nuclear Information System (INIS)

Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming

2016-01-01

The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells

Energy Technology Data Exchange (ETDEWEB)

Gao, Hui; Lin, Lu; Haq, Ihtesham Ul; Zeng, Shen-ming, E-mail: zengshenming@gmail.com

2016-04-22

The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development. - Highlights: • FSH inhibits the activation of NF-κB in porcine primary granulosa cells. • Inhibition of NF-κB by FSH promotes autophagy via JNK signaling in granulosa cells. • Increased autophagy contributes to progesterone production in granulosa cells. • This is the first report against beclin1 regulation in porcine granulosa cells.

Genetic Characterization of porcine circovirus type 2 isolated from different pig-farms in Croatia

DEFF Research Database (Denmark)

Rudan, Nevenka; Hjulsager, Charlotte Kristiane; Dupont, Kitt

2009-01-01

Histopathological fifi ndings in 25 pig tissue samples, which indicated PCVD (porcine circovirus diseases), were studied. Pig tissue samples originated from 5 different pig-farms in the north-west part of Croatia. Histopathological lesions showed two clinical pictures of the disease: porcine...

Topical timolol with and without benzalkonium chloride: epithelial permeability and autofluorescence of the cornea in glaucoma.

Science.gov (United States)

de Jong, C; Stolwijk, T; Kuppens, E; de Keizer, R; van Best, J

1994-04-01

Epithelial permeability and autofluorescence of the cornea were determined by fluorophotometry in 21 patients with open-angle glaucoma or ocular hypertension using timolol medication with the preservative benzalkonium chloride (BAC) and 2 weeks after changing to timolol medication without BAC. The investigation was performed to determine whether removal of BAC would reduce toxic effects on the cornea and complaints of sensations of burning or dry eye. Corneal epithelial permeability decreased significantly after changing medication (mean decrease per patient 27%, P = 0.025). Corneal autofluorescence increased significantly after changing medication suggesting an alteration in corneal metabolism (mean increase per patient 6%, P = 0.003). Timolol without BAC was found to be as effective as timolol with BAC in reducing intraocular pressure (P = 0.4). Removal of BAC from timolol resulted in an improvement of corneal epithelial barrier function and in a reduction of complaints. The improvement was found to be proportional to the duration of the preceding BAC-containing therapy.

Porcine head response to blast.

Science.gov (United States)

Shridharani, Jay K; Wood, Garrett W; Panzer, Matthew B; Capehart, Bruce P; Nyein, Michelle K; Radovitzky, Raul A; Bass, Cameron R 'dale'

2012-01-01

Recent studies have shown an increase in the frequency of traumatic brain injuries related to blast exposure. However, the mechanisms that cause blast neurotrauma are unknown. Blast neurotrauma research using computational models has been one method to elucidate that response of the brain in blast, and to identify possible mechanical correlates of injury. However, model validation against experimental data is required to ensure that the model output is representative of in vivo biomechanical response. This study exposes porcine subjects to primary blast overpressures generated using a compressed-gas shock tube. Shock tube blasts were directed to the unprotected head of each animal while the lungs and thorax were protected using ballistic protective vests similar to those employed in theater. The test conditions ranged from 110 to 740 kPa peak incident overpressure with scaled durations from 1.3 to 6.9 ms and correspond approximately with a 50% injury risk for brain bleeding and apnea in a ferret model scaled to porcine exposure. Instrumentation was placed on the porcine head to measure bulk acceleration, pressure at the surface of the head, and pressure inside the cranial cavity. Immediately after the blast, 5 of the 20 animals tested were apneic. Three subjects recovered without intervention within 30 s and the remaining two recovered within 8 min following respiratory assistance and administration of the respiratory stimulant doxapram. Gross examination of the brain revealed no indication of bleeding. Intracranial pressures ranged from 80 to 390 kPa as a result of the blast and were notably lower than the shock tube reflected pressures of 300-2830 kPa, indicating pressure attenuation by the skull up to a factor of 8.4. Peak head accelerations were measured from 385 to 3845 G's and were well correlated with peak incident overpressure (R(2) = 0.90). One SD corridors for the surface pressure, intracranial pressure (ICP), and head acceleration are

Decellularized human amniotic membrane: more is needed for an efficient dressing for protection of burns against antibiotic-resistant bacteria isolated from burn patients.

Science.gov (United States)

Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A

2015-11-01

Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

Science.gov (United States)

Pegels, N; González, I; Fernández, S; García, T; Martín, R

2012-01-01

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

Femtosecond lasers for microsurgery of cornea

International Nuclear Information System (INIS)

Vartapetov, Sergei K; Khudyakov, D V; Lapshin, Konstantin E; Obidin, Aleksei Z; Shcherbakov, Ivan A

2012-01-01

The review of femtosecond laser installations for medical applications is given and a new femtosecond ophthalmologic system for creation of a flap of corneal tissue during the LASIK operation is described. An all-fibre femtosecond laser emitting ∼400-fs pulses at 1067 nm is used. The pulse repetition rate can vary from 200 kHz up to 1 MHz. The output energy of the femtosecond system does not exceed 1 μJ. A specially developed objective with small spherical and chromatic aberrations is applied to focus laser radiation to an area of an eye cornea. The size of the focusing spot does not exceed 3 μm. To process the required area, scanning by a laser beam is applied with a speed no less than 5 m s -1 . At a stage of preliminary tests of the system, the Κ8 glass, organic PMMA glass and specially prepared agarose gels are used as a phantom of an eye. The femtosecond system is successfully clinically tested on a plenty of eyes of a pig and on several human eyes. The duration of the procedure of creation of a corneal flap does not exceed 20 s.

Femtosecond lasers for microsurgery of cornea

Energy Technology Data Exchange (ETDEWEB)

Vartapetov, Sergei K; Khudyakov, D V; Lapshin, Konstantin E; Obidin, Aleksei Z; Shcherbakov, Ivan A

2012-03-31

The review of femtosecond laser installations for medical applications is given and a new femtosecond ophthalmologic system for creation of a flap of corneal tissue during the LASIK operation is described. An all-fibre femtosecond laser emitting {approx}400-fs pulses at 1067 nm is used. The pulse repetition rate can vary from 200 kHz up to 1 MHz. The output energy of the femtosecond system does not exceed 1 {mu}J. A specially developed objective with small spherical and chromatic aberrations is applied to focus laser radiation to an area of an eye cornea. The size of the focusing spot does not exceed 3 {mu}m. To process the required area, scanning by a laser beam is applied with a speed no less than 5 m s{sup -1}. At a stage of preliminary tests of the system, the {Kappa}8 glass, organic PMMA glass and specially prepared agarose gels are used as a phantom of an eye. The femtosecond system is successfully clinically tested on a plenty of eyes of a pig and on several human eyes. The duration of the procedure of creation of a corneal flap does not exceed 20 s.

Ascorbic acid as a free radical scavenger in porcine and bovine aqueous humour.

Science.gov (United States)

Erb, Carl; Nau-Staudt, Kerstin; Flammer, Josef; Nau, Werner

2004-01-01

To study the antioxidant activity, UV absorption, concentration and stability of ascorbic acid (AA) in porcine and bovine aqueous humour (AH). Porcine and bovine AH was taken within 5 min after death and frozen at -70 degrees C. The characteristic UV absorption band of AA and the concentration of AA in AH was determined by UV spectrophotometry. The antioxidant activity of AA to serve as a free radical scavenger in AH has been determined by using a novel fluorescent probe for antioxidants, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO). The fluorescence lifetime and intensity of this probe reflect the concentration of dissolved antioxidants. The time-resolved fluorescence of DBO (laser excitation at 351 nm) in AH and in a neutral phosphate-buffered saline (PBS) solution containing only the natural amount of AA as an additive were measured. The characteristic UV absorption band of AA has its maximum at 266 nm in AH. The concentration of AA in porcine and bovine AH was found to be 0.547 +/- 0.044 and 1.09 +/- 0.16 mM, respectively, by spectrophotometry. The fluorescence lifetime of the probe DBO was reduced from 320 +/- 5 ns in pure aerated PBS to 205 +/- 5 ns in porcine AH and 165 +/- 3 ns in bovine AH. A detailed kinetic analysis of the lifetime shortening suggests that AA contributes approximately 75 and 85% to the antioxidant activity of porcine and bovine AH, respectively. Our experiments suggest that AA is the major contributor to the antioxidant activity of porcine and bovine AH. The role of AA to serve as an antioxidant in AH is discussed. In addition, UV spectrophotometry is established as an alternative method to determine the concentration of AA in AH. Copyright 2004 S. Karger AG, Basel

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

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Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

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Yamakoshi Fumiko

2011-02-01

Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

First identification of porcine parvovirus 7 in China.

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Xing, Xiulin; Zhou, Han; Tong, Ling; Chen, Yao; Sun, Yankuo; Wang, Heng; Zhang, Guihong

2018-01-01

Porcine parvovirus (PPV) are small, non-enveloped and single-stranded DNA viruses, taxonomically classifiable within the family Parvoviridae. Seven PPV genotypes (PPV1 to PPV7) have been identified to date. PPV7, the most recently discovered PPV genotype, was first reported in US pigs in 2016. To explore PPV7 status in Chinese pig populations a total of 64 serum samples collected from two commercial farms in Guangdong province in 2014 were analyzed. PPV7 DNA was detected in 32.8% (21/64) of tested samples. On the porcine circovirus type 2 (PCV2) positive farm, the prevalence rate of PPV7 was 65.5% (19/29) which was significantly higher than that on the PCV2 negative farm (2/35, 5.7%), indicating a possible association between PCV2 and PPV7 infections. The sequences of three PPV7 strains were determined. Phylogenetic analysis revealed that the identified PPV7 strains circulating in China shared 98.7%-99.7% nucleotide homology with the US strain. Further sequence comparison analysis indicated that GD-2014-2 and GD-2014-3 possess a consecutive 9-nt deletion in the VP gene. This is the first report of the existence of PPV7 in China and this finding will strengthen understanding of the epidemiology of porcine parvovirus in Chinese pigs.

Perspectives on the Evolution of Porcine Parvovirus.

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Oh, Woo-Taek; Kim, Ri-Yeon; Nguyen, Van-Giap; Chung, Hee-Chun; Park, Bong-Kyun

2017-07-26

Porcine parvovirus (PPV) is one of the main causes of porcine reproductive failure. It is important for swine industries to understand the recent trends in PPV evolution. Previous data show that PPV has two genetic lineages originating in Germany. In this study, two more genetic lineages were defined, one of which was distinctly Asian. Additionally, amino acid substitutions in European strains and Asian strains showed distinct differences in several regions of the VP2 gene. The VP1 gene of the recent PPV isolate (T142_South Korea) was identical to that of Kresse strain isolated in the USA in 1985, indicating that modern PPV strains now resemble the original strains (Kresse and NADL-2). In this study, we compared strains isolated in the 20th century to recent isolates and confirmed the trend that modern strains are becoming more similar to previous strains.

Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor

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Huanzhong Cui

2016-10-01

Full Text Available A series of antagonists specifically targeting growth hormone receptors (GHR in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH. Therefore, in this study, we developed and characterized a porcine GHR (pGHR antibody antagonist (denoted by AN98 via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1 secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

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Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro

2013-12-01

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immobilization of pseudorabies virus in porcine tracheal respiratory mucus revealed by single particle tracking.

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Xiaoyun Yang

Full Text Available Pseudorabies virus (PRV initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: -31.8±1.5 mV experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (-49.8±0.6 mV and positively (36.7±1.1 mV charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (-9.6±0.8 mV diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV

Stoichiometric iodination and purification of porcine insulin with chloramine T for radioimmunoassay

International Nuclear Information System (INIS)

Toledo e Souza, I.T. de; Giannella Neto, D.; Wajchenberg, B.L.

1986-01-01

Stoichiometric iodination and purification of porcine insulin was performed to the general method of Hunter and Greenwood (classical chloramine T) with modifications recommended by Roth (chloramine T is added in limiting amounts in multiple small additions). Satisfactory specific activity of the labeled hormone was obtained and the characteristics of the radioimmunoassay, based on the competition of the 125-I labeled porcine and cold insulin for specific antibody were studied. (Author) [pt

Mechanics of fresh, frozen-thawed and heated porcine liver tissue.

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Wex, Cora; Stoll, Anke; Fröhlich, Marlen; Arndt, Susann; Lippert, Hans

2014-06-01

For a better understanding of the effects of thermally altered soft tissue, the biothermomechanics of these tissues need to be studied. Without the knowledge of the underlying physical processes and the parameters that can be controlled clinically, thermal treatment of cancerous hepatic tissue or the preservation of liver grafts are based primarily on trial and error. Thus, this study is concerned with the investigation of the influence of temperature on the rheological properties and the histological properties of porcine liver. Heating previously cooled porcine liver tissue above 40 °C leads to significant, irreversible stiffness changes observed in the amplitude sweep. The increase of the complex shear module of healthy porcine liver from room temperature to 70 °C is approximately 9-fold. Comparing the temperatures -20 °C and 20 °C, no significant difference of the mechanical properties was observed. Furthermore, there is a strong relation between the mechanical and histological properties of the porcine liver. Temperatures above 40 °C destroy the collagen matrix within the liver tissue. This results in the alteration of the biomechanical properties. The time-temperature superposition principle is applied to generate temperature-dependent shift factors that can be described by a two-part exponential function model with an inflection temperature of 45 °C. Tumor ablation techniques such as heating or freezing have a significant influence on the histology of liver tissue. However, only for temperatures above body temperature an influence on the mechanical properties of hepatic tissues was noticeable. Freezing up to -20 °C did not affect the liver mechanics.

Characterization of porcine MMP-2 and its association with immune traits

DEFF Research Database (Denmark)

Huang, Honggang; Zhao, Weimin; Tang, Zhonglin

2009-01-01

cloned the 5'-upstream sequence, 3'-downstream sequence as well as other missed genomic sequences of porcine MMP-2, the genomic structure and the promotor sequence were analyzed and found to share high similarity with those of human MMP-2. Porcine MMP-2 was assigned to SSC6p14-p15, and closely linked......Matrix metalloproteinase-2 (MMP-2) plays important roles in inflammation and immunity besides its basic role in degrading and remodelling extracellular matrix (ECM). The expression of MMP-2 is up-regulated in many human as well as animal models of inflammatory and immune diseases. In this study, we...... to microsatellite SW1108 (53cR, LOD score 7.59) by IMpRH panel. Real-time PCR analysis revealed that the expression of porcine MMP-2 was remarkably different in diverse tissues, a high level expression was observed in the testis and uterus, relatively low expression in other tissues. Allele frequencies...

An in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome.

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Dawson, Harry D; Smith, Allen D; Chen, Celine; Urban, Joseph F

2017-04-01

Emerging evidence suggests that swine are a scientifically acceptable intermediate species between rodents and humans to model immune function relevant to humans. The swine genome has recently been sequenced and several preliminary structural and functional analysis of the porcine immunome have been published. Herein we provide an expanded in silico analysis using an improved assembly of the porcine transcriptome that provides an in depth analysis of genes that are related to inflammasomes, responses to Toll-like receptor ligands, and M1 macrophage polarization and Escherichia coli as a model organism. Comparisons of the expansion or contraction of orthologous gene families indicated more similar rates and classes of genes in humans and pigs than in mice; however several novel porcine or artiodactyl-specific paralogs or pseudogenes were identified. Conservation of homology and structural motifs of orthologs revealed that the overall similarity to human proteins was significantly higher for pigs compared to mouse. Despite these similarities, two out of four canonical inflammasome pathways, Absent in melanoma 2 (AIM2) and NLR family and CARD domain containing 4 (NLRC4), were found to be missing in pigs. Pig M1 Mφ polarization in response to interferon-γ (IFN-γ) and lipopolysaccharide (LPS) was assessed, via the transcriptome, using next generation sequencing. Our analysis revealed predominantly human-like responses however some, mouse-like responses were observed, as well as induction of numerous pig or artiodactyl-specific genes. This work supports using swine to model both human immunological and inflammatory responses to infection. However, caution must be exercised as pigs differ from humans in several fundamental pathways. Published by Elsevier B.V.

Xanthelasma palpebrarum with arcus cornea: A clinical and biochemical study

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Pragya Ashok Nair

2016-01-01

Full Text Available Background: Xanthelasma palpebrarum (XP is characterized by sharply demarcated yellowish flat plaques on upper and lower eyelids. It is commonly seen in women with a peak incidence at 30–50 years. It is also considered as the cutaneous marker of underlying atherosclerosis along with the disturbed lipid metabolism. XP and corneal arcus are associated with increased levels of serum cholesterol and low-density lipoprotein (LDL cholesterol. Aims and Objectives: To study the clinical pattern of XP, its relationship with lipid profile and association with arcus cornea. Materials and Methods: This study was conducted at Department of Dermatology and Opthalmology, between August 2013 and January 2015. Patients with clinical diagnosis of XP who visited skin outpatient department and willing to undergo lipid profile test and eye examination were included in the study. Data regarding demographics, clinical findings, family history, and past history were noted along with the lipid profile details. Data of age-matched healthy controls were taken for comparison. The clinical profile of the participants was presented using frequency and proportions. Gender wise analysis comparing the lipid profile in cases with XP and without XP was done using independent sample t-test. Results: Total 49 patients of XP, 81.6% were females. Maximum, 35% patients were among 50–60 years of age and 69.4% were homemakers by occupation. The average lipid values were-cholesterol 210.57 mg%, triglyceride 123.06 mg%. LDL 142.79 mg% and VLDL 30.95 mg% among patients of XP. Arcus cornea was found in 20% cases of XP. Conclusions: Patients of XP requires proper investigation at the onset and regular follow-up thereafter for any altered lipid profile and early diagnosis of coronary artery disease.

Partial Denervation of Subbasal Axons Persists Following Debridement Wounds to the Mouse Cornea

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Pajoohesh-Ganji, Ahdeah; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Kyne, Briana M.; Saban, Daniel R.; Stepp, Mary Ann

2015-01-01

Although sensory reinnervation occurs after injury in the PNS, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify subbasal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of subbasal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7d after superficial trephination, subbasal axon density returns to control levels; by 28d the vortex reforms. Although axon density is similar to control 14d after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14d, axons retract from the center leaving the subbasal axon density reduced by 37.2% and 36.8% at 28d after dulled blade and rotating burr wounding, respectively, compared to control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration associated genes (RAGs) involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7d after injury and by 14d and 28d after wounding, many of these basal cells undergo apoptosis and die. While subbasal axons are restored to their normal density and morphology after superficial trephination, subbasal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14d

Porcine Is a Positional Candidate Gene Associated with Growth and Fat Deposition

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Bong Hwan Choi

2012-12-01

Full Text Available Crosses between Korean and Landrace pigs have revealed a large quantitative trait loci (QTL region for fat deposition in a region (89 cM of porcine chromosome 4 (SSC4. To more finely map this QTL region and identify candidate genes for this trait, comparative mapping of pig and human chromosomes was performed in the present study. A region in the human genome that corresponds to the porcine QTL region was identified in HSA1q21. Furthermore, the LMNA gene, which is tightly associated with fat augmentation in humans, was localized to this region. Radiation hybrid (RH mapping using a Sus scrofa RH panel localized LMNA to a region of 90.3 cM in the porcine genome, distinct from microsatellite marker S0214 (87.3 cM. Two-point analysis showed that LMNA was linked to S0214, SW1996, and S0073 on SSC4 with logarithm (base 10 of odds scores of 20.98, 17.78, and 16.73, respectively. To clone the porcine LMNA gene and to delineate the genomic structure and sequences, including the 3′untranslated region (UTR, rapid amplification of cDNA ends was performed. The coding sequence of porcine LMNA consisted of 1,719 bp, flanked by a 5’UTR and a 3’UTR. Two synonymous single nucleotide polymorphisms (SNPs were identified in exons 3 and 7. Association tests showed that the SNP located in exon 3 (A193A was significantly associated with weight at 30 wks (p<0.01 and crude fat content (p<0.05. This association suggests that SNPs located in LMNA could be used for marker-assisted selection in pigs.

High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

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Ying Ao

Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

Oxidative Stress to the Cornea, Changes in Corneal Optical Properties, and Advances in Treatment of Corneal Oxidative Injuries

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Cestmir Cejka

2015-01-01

Full Text Available Oxidative stress is involved in many ocular diseases and injuries. The imbalance between oxidants and antioxidants in favour of oxidants (oxidative stress leads to the damage and may be highly involved in ocular aging processes. The anterior eye segment and mainly the cornea are directly exposed to noxae of external environment, such as air pollution, radiation, cigarette smoke, vapors or gases from household cleaning products, chemical burns from splashes of industrial chemicals, and danger from potential oxidative damage evoked by them. Oxidative stress may initiate or develop ocular injury resulting in decreased visual acuity or even vision loss. The role of oxidative stress in the pathogenesis of ocular diseases with particular attention to oxidative stress in the cornea and changes in corneal optical properties are discussed. Advances in the treatment of corneal oxidative injuries or diseases are shown.

Porcine prion protein amyloid.

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Hammarström, Per; Nyström, Sofie

2015-01-01

Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

Penetration of 45Ca in rabbit cornea following the introduction of air in the anterior chamber and its paracenthesis and following experimental intraocular hypertension

International Nuclear Information System (INIS)

Votockova, J.; Obenberger, J.; Babicky, A.

1977-01-01

In one group of Chinchilla rabbits the aqueous humor was withdrawn and the anterior chamber was refilled with air; in a second group only simple paracenthesis and withdrawal were performed; in a third group of animals experimental intraocular hypertension was produced by means of a subconjunctival injection of hypertonic sodium chloride. Immediately after these operations, a solution of 45 CaCl 2 was intravenously injected, and the rabbits were sacrificed after an interval of 30 min or 120 min. The corneas were excised and the radioactivity in central parts (trephined discs of 8 mm in diameter) and in remaining peripheral parts was determined. In all groups the corneal radioactivity in operated eyes exceeded the values found in the contralateral corneas of not operated eyes, radioactivity in these eyes being equal to the radioactivity found in the corneas of not operated rabbits in the control group. Various possible explanations of the increase of corneal radioactivity in operated eyes are briefly discussed. (author)

Effect of a pulsed magnetic field on permeability of the cornea and sorption properties of the tissue structures and refractive media of the eye

International Nuclear Information System (INIS)

Skrinnik, A.V.

1986-01-01

The author attempts to obtain direct proof of increased permeability of the cornea and enhanced sorption properties of the tissue structures and refractive media of the eye under the influence of a pulsed magnetic field (MF). In the course of the investigation, the method of radioactive indication of two substances ( 35 S-streptomycin and 75 Se-methionine), widely used in opthalmologic practice, was used. The radioactivity of the working solution of the preparations was 300,000 cpm in 0.1 ml. Changes in the permeability of the cornea were assessed on the basis of changes in radioactivity of the aqueous humor. The concentration of 35 S-streptomycin in aqueous humor of the animals immediately after exposure to MF was significantly higher than in the control. Analysis of permeability of the cornea for 75 Se-methionine also showed increased penetration of radioactivity into the aqueous. The results are evidence of the greater penetrating power of 75 Se-methionine

Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development

DEFF Research Database (Denmark)

Venø, Morten T; Hansen, Thomas B; Venø, Susanne T

2015-01-01

BACKGROUND: Recently, thousands of circular RNAs (circRNAs) have been discovered in various tissues and cell types from human, mouse, fruit fly and nematodes. However, expression of circRNAs across mammalian brain development has never been examined. RESULTS: Here we profile the expression of circ......RNA in five brain tissues at up to six time-points during fetal porcine development, constituting the first report of circRNA in the brain development of a large animal. An unbiased analysis reveals a highly complex regulation pattern of thousands of circular RNAs, with a distinct spatio-temporal expression...... are functionally conserved between mouse and human. Furthermore, we observe that "hot-spot" genes produce multiple circRNA isoforms, which are often differentially expressed across porcine brain development. A global comparison of porcine circRNAs reveals that introns flanking circularized exons are longer than...

Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

Science.gov (United States)

2017-02-01

a cell population particularly suitable for low serum propagation, provided that appropriate growth factors are available. A low serum medium...of MGK. 15. SUBJECT TERMS Cornea, chemical warfare agent, corneal endothelial cell, endothelium, growth , isolation, mouse, rabbit, porcine, in...with corneal SM exposure.2 A primary requirement in achieving this goal is to develop methods that enable the isolation of a pure CEC population and

In vivo survival of [14C]sucrose-loaded porcine carrier erythrocytes

International Nuclear Information System (INIS)

DeLoach, J.R.

1983-01-01

Porcine carrier erythrocyte survival was measured in adult pigs. [14C]Sucrose-loaded erythrocytes had a biphasic survival curve, with as much as 50% of the cells removed from circulation in the first 24 hours. The remaining cells had a 35-day half-life. Encapsulation values were measured for porcine erythrocytes and entrapment of [14C]sucrose was greater than 45%. Addition of inosine and glucose to the dialyzed cells and to the final wash buffer before reinjection of autologous cells did not improve their survival

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

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K.C. Médici

2010-01-01

Full Text Available Group B rotaviruses (RV-B were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8 were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

DEFF Research Database (Denmark)

Valleh, Mehdi Vafaye; Zandi, Nahid Karimi; Rasmussen, Mikkel Aabech

2017-01-01

It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the opti......It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action...... of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus-oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0-100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined...

Experience of Comamonas Acidovorans Keratitis with Delayed Onset and Treatment Response in Immunocompromised Cornea

OpenAIRE

Lee, Sang Mok; Kim, Mee Kum; Lee, Jae Lim; Wee, Won Ryang; Lee, Jin Hak

2008-01-01

Purpose To report 2 cases of Comamonas Acidovorans keratitis in immunocompromised cornea. Methods A complete review of the medical records of the two cases of Comamonas acidovorans keratitis. Results We found some similarities in clinical courses of two cases. Both of them showed development of keratitis during the management with corticosteroids, delayed onset, slow response to antibiotics, and relatively less affected corneal epithelium. Conclusions Comamonas Acidovorans is known as a less ...

Interdigitated electrode (IDE) for porcine detection based on titanium dioxide (TiO{sub 2}) thin films

Energy Technology Data Exchange (ETDEWEB)

Nordin, N.; Azizah, N. [Institute of Electronic Engineering (INEE), Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia); Hashim, U., E-mail: uda@unimap.edu.my [Institute of Electronic Engineering (INEE), Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia); School of Microelctronic Engineering, Universiti Malaysia Perlis, 01000 Kangar Perlis (Malaysia)

2016-07-06

Interdigited Electrode (IDE) porcine detection can be accomplished to authenticate the halal issue that has been a concern to Muslim not only in Malaysia but all around the world. The method used is photolithography that used the p-type photoresist on the spin coater with 2500 rpm. Bare IDEs device is deposited with Titanium Dioxide (TiO{sub 2}) to improve the performance of the device. The result indicates that current-voltage (I-V) measurement of porcine probe line slightly above porcine target due to negative charges repelled each other. The IDE device can detect the porcine presence in food as lowest as 1.0 µM. Better performance of the device can be achieved with the replacement of gold deposited to trigger more sensitivity of the device.

Effect of biometric characteristics on biomechanical properties of the cornea in cataract patient.

Science.gov (United States)

Song, Xue-Fei; Langenbucher, Achim; Gatzioufas, Zisis; Seitz, Berthold; El-Husseiny, Moatasem

2016-01-01

To determine the impact of biometric characteristics on the biomechanical properties of the human cornea using the ocular response analyzer (ORA) and standard comprehensive ophthalmic examinations before and after standard phacoemulsification. This study comprised 54 eyes with cataract with significant lens opacification in stages I or II that underwent phacoemulsification (2.8 mm incision). Corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg), and corneal-compensated intraocular pressure (IOPcc) were measured by ORA preoperatively and at 1mo postoperatively. Biometric characteristics were derived from corneal topography [TMS-5, anterior equivalent (EQTMS) and cylindric (CYLTMS) power], corneal tomography [Casia, anterior and posterior equivalent (EQaCASIC, EQpCASIA) and cylindric (CYLaCASIA, CYLpCASIA) power], keratometry [IOLMaster, anterior equivalent (EQIOL) and cylindric (CYLIOL) power] and autorefractor [anterior equivalent (EQAR)]. Results from ORA were analyzed and correlated with those from all other examinations taken at the same time point. Preoperatively, CH correlated with EQpCASIA and CYLpCASIA only (P=0.001, P=0.002). Postoperatively, IOPg and IOPcc correlated with all equivalent powers (EQTMS, EQIOL, EQAR, EQaCASIA and EQpCASIA) (P=0.001, P=0.007, P=0.001, P=0.015, P=0.03 for IOPg and PBiometric characteristics may significantly affect biomechanical properties of the cornea in terms of CH, IOPcc and IOPg before, but even more after cataract surgery.

Porcine Tricuspid Valve Anatomy and Human Compatibility

DEFF Research Database (Denmark)

Waziri, Farhad; Lyager Nielsen, Sten; Hasenkam, J. Michael

2016-01-01

before clinical use. The study aim was to evaluate and compare the tricuspid valve anatomy of porcine and human hearts. METHODS: The anatomy of the tricuspid valve and the surrounding structures that affect the valve during a cardiac cycle were examined in detail in 100 fresh and 19 formalin...

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

DEFF Research Database (Denmark)

Kvisgaard, Lise Kirstine

This PhD thesis presents the diversity of Porcine Reproductive and Respiratory Syndrome viruses (PRRSV) circulating in the Danish pig population. PRRS is a disease in pigs caused by the PRRS virus resulting in reproductive failures in sows and gilts and respiratory diseases in pigs . Due to genetic...

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Kamerling, J.P.; Rijkse, I.; Maas, A.A.M.; Kuik, J.A. van

1988-01-01

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on

Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium.

Directory of Open Access Journals (Sweden)

Silke S Zakrzewski

Full Text Available Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS or species-specific (porcine serum, PS conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS, compared to conventional FBS culture (IPEC-J2/FBS, the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.

A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis.

Science.gov (United States)

Cortina, María E; Balzano, Rodrigo E; Rey Serantes, Diego A; Caillava, Ana J; Elena, Sebastián; Ferreira, A C; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

2016-06-01

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice.

Science.gov (United States)

Alexander, George; Carlsen, Harald; Blomhoff, Rune

2006-04-01

To determine the dynamics of Nuclear Factor-kappaB (NF-kappaB) in murine corneal pathology and the role of NF-kappaB in maintaining corneal clarity after ultraviolet B radiation insult, transgenic mice containing NF-kappaB-luciferase reporter were exposed to LPS (bacterial lipopolysaccharide), TNF-alpha (Tumor Necrosis Factor-alpha) or 4 kJ m(-2) UV-B radiation. NF-kappaB decoy oligonucleotides were also administered in some of the UV-B experiments. Following various exposure times, the mice were sacrificed and whole eyes or corneal tissues were obtained. Whole eyes were examined for scattering using a point-source optical imaging technique. Tissue homogenates were examined for luciferase activity using a luminometer. TNF-alpha and LPS-injected NF-kappaB-luciferase transgenic mice demonstrated 3-10-fold increases in cornea NF-kappaB with peak activities at 4 and 6 hr post-injection, respectively. Mice exposed to 4 kJ m(-2) UV-B exhibited a 3-fold increase in NF-kappaB activity 4 hr post-exposure. The administration of NF-kappaB-decoy oligonucleotides to mice had the effect of reducing UV-B-induced NF-kappaB activity in the cornea and significantly increasing the amount of light scattering in UV-B exposed corneas 7 days post-UV-B exposure when compared to sham injected mice. These results indicate that NF-kappaB is activated in cornea in pathologies that involves increased plasma levels of LPS and TNF-alpha, as well as direct UV-B exposure, and suggest that NF-kappaB activation play an essential part in the corneal healing process.

Case report: a novel KERA mutation associated with cornea plana and its predicted effect on protein function

DEFF Research Database (Denmark)

Roos, Laura; Bertelsen, Birgitte; Harris, Pernille

2015-01-01

of the keratocan gene (KERA) on chromosome 12q22. To date, only nine different disease-associated KERA mutations, including four missense mutations, have been described. Case presentation: In this report, we present clinical data from a Turkish family with autosomal recessive cornea plana. In some of the affected...

Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

Science.gov (United States)

Schneeberger, M; Brodard, I; Overesch, G

2015-04-02

Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4

Thermal lens measurements in the cornea.

Science.gov (United States)

Venkatesh, S; Guthrie, S; Cruickshank, F R; Bailey, R T; Foulds, W S; Lee, W R

1985-02-01

Q-switched pulses from a neodymium/YAG (yttrium-aluminium-garnet) laser were passed through corneal discs taken from the enucleated eyes of three baboons and four rabbits. The time course of heat dissipation following absorption of laser energy by the tissue was studied with the use of a second continuous wave laser beam acting as a probe. It was found that the absorption of each neodymium/YAG pulse created a transient divergent lens within the cornea as theoretical considerations predicted. The relaxation time that characterised the decay of this thermal lens for a 1/e laser beam diameter of 2.0 mm was found to be 2.3 +/- 0.1 s (mean +/- standard error for 12 separate groups of measurements). Our results show that Q-switched laser pulses passing through apparently unaffected transparent tissues can induce thermal lens effects which persist for several seconds. The optical transfer of each pulse in a stream will be identical only if enough time is left between pulses for the tissues to return to their initial state. Therefore, when such laser pulses sharply focused to perform high precision intraocular surgery are used, thermal lensing in the transparent ocular media must limit the rate at which pulses can be usefully delivered.

Physicochemical characterization of porcine bone-derived grafting material and comparison with bovine xenografts for dental applications.

Science.gov (United States)

Lee, Jung Heon; Yi, Gyu Sung; Lee, Jin Woong; Kim, Deug Joong

2017-12-01

The physicochemical properties of a xenograft are very important because they strongly influence the bone regeneration capabilities of the graft material. Even though porcine xenografts have many advantages, only a few porcine xenografts are commercially available, and most of their physicochemical characteristics have yet to be reported. Thus, in this work we aimed to investigate the physicochemical characteristics of a porcine bone grafting material and compare them with those of 2 commercially available bovine xenografts to assess the potential of xenogenic porcine bone graft materials for dental applications. We used various characterization techniques, such as scanning electron microscopy, the Brunauer-Emmett-Teller adsorption method, atomic force microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and others, to compare the physicochemical properties of xenografts of different origins. The porcine bone grafting material had relatively high porosity (78.4%) and a large average specific surface area (SSA; 69.9 m 2 /g), with high surface roughness (10-point average roughness, 4.47 µm) and sub-100-nm hydroxyapatite crystals on the surface. Moreover, this material presented a significant fraction of sub-100-nm pores, with negligible amounts of residual organic substances. Apart from some minor differences, the overall characteristics of the porcine bone grafting material were very similar to those of one of the bovine bone grafting material. However, many of these morphostructural properties were significantly different from the other bovine bone grafting material, which exhibited relatively smooth surface morphology with a porosity of 62.0% and an average SSA of 0.5 m 2 /g. Considering that both bovine bone grafting materials have been successfully used in oral surgery applications in the last few decades, this work shows that the porcine-derived grafting material possesses most of the key physiochemical characteristics required for its

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication.

Science.gov (United States)

Demirhan, Yasemin; Ulca, Pelin; Senyuva, Hamide Z

2012-03-01

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cardiac Dysfunction in a Porcine Model of Pediatric Malnutrition

DEFF Research Database (Denmark)

Fabiansen, Christian; Lykke, Mikkel; Hother, Anne-Louise

2015-01-01

BACKGROUND: Half a million children die annually of severe acute malnutrition and cardiac dysfunction may contribute to the mortality. However, cardiac function remains poorly examined in cases of severe acute malnutrition. OBJECTIVE: To determine malnutrition-induced echocardiographic disturbances...... and longitudinal changes in plasma pro-atrial natriuretic peptide and cardiac troponin-T in a pediatric porcine model. METHODS AND RESULTS: Five-week old piglets (Duroc-x-Danish Landrace-x-Yorkshire) were fed a nutritionally inadequate maize-flour diet to induce malnutrition (MAIZE, n = 12) or a reference diet...... groups. The myocardial performance index was 86% higher in MAIZE vs AGE-REF (pMalnutrition associates with cardiac dysfunction in a pediatric porcine model by increased myocardial performance index and pro-atrial natriuretic peptide...

Porcine Circovirus Diseases: A review of PMWS

DEFF Research Database (Denmark)

Baekbo, P.; Kristensen, C. S.; Larsen, L. E.

2012-01-01

Porcine Circo Virus type 2 have been coming on the market and many studies have shown great benefits of these to control PMWS. Today, sow vaccines as well as piglet vaccines are available in most countries. An extensive meta‐analysis of many of the vaccines has shown a comparable good efficacy...

A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model▿

OpenAIRE

Opriessnig, T.; Shen, H. G.; Pal, N.; Ramamoorthy, S.; Huang, Y. W.; Lager, K. M.; Beach, N. M.; Halbur, P. G.; Meng, X. J.

2011-01-01

The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine p...

Clinical characteristics of keratoconus patients in Malaysia: a review from a cornea specialist centre

OpenAIRE

Mohd-Ali, Bariah; Abdu, Mustafa; Yaw, Choong Yean; Mohidin, Norhani

2012-01-01

Purpose: To evaluate the demographic profile, refraction, visual acuity (VA), corneal curvature (K) and severity of keratoconus (KC) patients from a cornea specialist centre in Malaysia. This may improve the management of KC patients in this country. Methods: Records of 13,000 patients were reviewed. The patients were categorized into 4 age groups: (Gp 1: 42 years). Refraction and VA were determined using subjective refraction and Snellen chart. Corneal curvature was measured using Orbscan...

Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

International Nuclear Information System (INIS)

Dumpala, Pradeep R.; Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A.; Parker, Thomas S.; Levine, Daniel M.; Smith, Barry H.; Gazda, Lawrence S.

2016-01-01

Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

Energy Technology Data Exchange (ETDEWEB)

Dumpala, Pradeep R., E-mail: pdumpala@rixd.org [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Parker, Thomas S.; Levine, Daniel M. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); Smith, Barry H. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); NewYork-Presbyterian Hospital, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States); Gazda, Lawrence S. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States)

2016-08-05

Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

Region-Specific Effect of the Decellularized Meniscus Extracellular Matrix on Mesenchymal Stem Cell-Based Meniscus Tissue Engineering.

Science.gov (United States)

Shimomura, Kazunori; Rothrauff, Benjamin B; Tuan, Rocky S

2017-03-01

The meniscus is the most commonly injured knee structure, and surgical repair is often ineffective. Tissue engineering-based repair or regeneration may provide a needed solution. Decellularized, tissue-derived extracellular matrices (ECMs) have received attention for their potential use as tissue-engineered scaffolds. In considering meniscus-derived ECMs (mECMs) for meniscus tissue engineering, it is noteworthy that the inner and outer regions of the meniscus have different structural and biochemical features, potentially directing the differentiation of cells toward region-specific phenotypes. To investigate the applicability of mECMs for meniscus tissue engineering by specifically comparing region-dependent effects of mECMs on 3-dimensional constructs seeded with human bone marrow mesenchymal stem cells (hBMSCs). Controlled laboratory study. Bovine menisci were divided into inner and outer halves and were minced, treated with Triton X-100 and DNase, and extracted with urea. Then, hBMSCs (1 × 10 6 cells/mL) were encapsulated in a photo-cross-linked 10% polyethylene glycol diacrylate scaffold containing mECMs (60 μg/mL) derived from either the inner or outer meniscus, with an ECM-free scaffold as a control. The cell-seeded constructs were cultured with chondrogenic medium containing recombinant human transforming growth factor β3 (TGF-β3) and were analyzed for expression of meniscus-associated genes as well as for the collagen (hydroxyproline) and glycosaminoglycan content as a function of time. Decellularization was verified by the absence of 4',6-diamidino-2-phenylindole (DAPI)-stained cell nuclei and a reduction in the DNA content. Quantitative real-time polymerase chain reaction showed that collagen type I expression was significantly higher in the outer mECM group than in the other groups, while collagen type II and aggrecan expression was highest in the inner mECM group. The collagen (hydroxyproline) content was highest in the outer mECM group, while the

In vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta

Directory of Open Access Journals (Sweden)

Kobayashi A

2014-02-01

Full Text Available Akira Kobayashi, Tomomi Higashide, Hideaki Yokogawa, Natsuko Yamazaki, Toshinori Masaki, Kazuhisa Sugiyama Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan Objective: To report the in vivo laser confocal microscopy findings of a cornea with osteogenesis imperfecta (OI with special attention to the abnormality of Bowman's layer and sub-Bowman's fibrous structures (K-structures. Patients and methods: Two patients (67-year-old male and his 26-year-old son with OI type I were included in this study. Slit lamp biomicroscopic and in vivo laser confocal microscopic examinations were performed for both patients. Central corneal thickness and central endothelial cell density were also measured. Results: Although the corneas looked clear with normal endothelial density for both eyes in both patients, they were quite thin (386 µm oculus dexter (OD (the right eye and 384 µm oculus sinister (OS (the left eye in the father and 430 µm OD and 425 µm OS in the son. In both patients, slit lamp biomicroscopic and in vivo laser confocal microscopic examination showed similar results. Anterior corneal mosaics produced by rubbing the eyelid under fluorescein were completely absent in both eyes. In vivo laser confocal microscopy revealed an absent or atrophic Bowman's layer; a trace of a presumed Bowman's layer and/or basement membrane was barely visible with high intensity. Additionally, K-structures were completely absent in both eyes. Conclusion: The absence of K-structures and fluorescein anterior corneal mosaics strongly suggested an abnormality of Bowman's layer in these OI patients. Keywords: osteogenesis imperfecta, K-structure, confocal microscopy, Bowman's layer

Factors influencing transmission of porcine cysticercosis in Tanzania

DEFF Research Database (Denmark)

Braae, Uffe Christian; Wendy, Harrison; Magnussen, Pascal

Understanding the factors contributing to the transmission of Taenia solium in sub-Saharan Africa is essential for control. This study aimed to elucidate factors concerning the transmission of porcine cysticercosis in an endemic area. A longitudinal study composed of three cross-sectional surveys...

Porcine circovirus: transcription and rolling-circle DNA replication

Science.gov (United States)

This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759-1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesi...

Substitution of porcine small intestinal submucosa for rabbit Achilles tendon, an experimental study.

Science.gov (United States)

Gu, Yan; Dai, Kerong

2002-09-25

To study the effect of substitution of porcine small intestinal submucosa (SIS) for rabbit Achilles tendon. Porcine SIS was taken out and processed. Part of Achilles tendons of 20 rabbits' right legs were removed and substituted by porcine SIS and the Achilles tendon of the left legs were used as controls. One, four, eight, twelve, and sixteen weeks after the operation 4 rabbits were killed and their right Achilles tendons were taken out to be examined histologically and their maximum load was tested. One week after the operation, the porcine SIS was already fused with the remaining part of rabbit Achilles tendon. Sixteen weeks after all the Achilles tendons looked like normal one. The maximum load of experimental Achilles tendon was 48 N +/- 9 N one week after the operation, and increased gradually. In the 16th week after the operation, the maximum load was 178 N +/- 6 N for the experimental Achilles tendon and 174 N +/- 10 N for the control tendon. The differences of maximum load between different weeks after operation, except that between one week and 4 weeks after, were statistically significant (P Achilles tendon is effective, thus proving the feasibility of in vivo tissue engineering technology.

Toward Development of Pluripotent Porcine Stem Cells by Road Mapping Early Embryonic Development

DEFF Research Database (Denmark)

Petkov, Stoyan; Freude, Kristine; Mashayekhi-Nezamabadi, Kaveh

2017-01-01

The lack in production of bona fide porcine pluripotent stem cells has definitely been hampered by a lack of research into porcine embryo development. Embryonic development in mammals is the extraordinary transition of a single-celled fertilized zygote into a complex fetus, which occurs...... in the uterus of the maternal adult during the early stages of gestation. Biomedical pig models could serve as genetic backgrounds for establishment of embryonic stem cells (ESCs) or other pluripotent stem cells (such as iPSC), which may be used to model and study diseases in vitro. This chapter provides...... insight into the current knowledge of pluripotent states in the developing pig embryo and the current status in establishment of bona fide porcine ESC (pESC) and piPSCs. It reflects the potential causes underlying the difficulty in establishing pluripotent stem cells and reviews recent data on global...

Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea.

Science.gov (United States)

Pajoohesh-Ganji, Ahdeah; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Kyne, Briana M; Saban, Daniel R; Stepp, Mary Ann

2015-11-01

Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal

Photorefractive Keratectomy (PRK) is Safe and Effective for Patients with Myopia and Thin Corneas.

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Naderi, Mostafa; Ghadamgahi, Saeed; Jadidi, Khosrow

2016-01-01

The aim of this study was to evaluate the long-term safety and efficacy of photorefractive keratectomy (PRK) for patients with myopia and thin corneas. In this retrospective case series, we included 74 eyes of 38 patients with myopia and central corneal thickness (CCT) PRK and had a mean postoperative follow-up period of four years. The following factors were evaluated: CCT, refraction, uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA), ablation depth, safety and efficacy indices (i.e., the ratio of the mean postoperative BCVA to the mean preoperative BCVA, and the ratio of the mean postoperative UCVA to mean preoperative the BCVA, respectively), and evidence of corneal ectasia (based on Orbscan topography images).The patients were aged 20 - 46 years (mean ±SD age, 28.18± 6.82 years). The mean ± SD pre- and postoperative CCTwas485.92 ± 9.27 µm and 434.84 ± 20.48 µm, respectively. The mean ± SD pre- and postoperative myopia was -2.77 D ± 1.51 and -0.24 ± 0.39 D, respectively, and the mean ± SD pre- and postoperative astigmatism was -0.82 D ± 0.99 and -0.37 ± 0.37 D, respectively. The mean pre- and postoperative BCVA and postoperative UCVA was 0.011 ± 0.03 Logarithm of the Minimum Angle of Resolution (log MAR), 0.003 ± 0.01 log MAR, and 0.054 ± 0.09 log MAR, respectively. The mean ± SD ablation depth, safety index and efficacy index was 54.34 ± 16.28 µm, 0.02 ± 0.12, and 0.11 ± 0.50, respectively. Regarding the postoperative corneal clarity, 72 eyes (97.3%) had a clear cornea (grade 0) and the remaining two eyes of one patient (2.70%) had a trace haze (grade 1). There was no evidence of corneal ectasia on any of the Orbscan topography images. Thus, among patients with myopia and thin corneas (PRK seems to be acceptable in terms of both safety and efficacy 4 years after surgery, based on the stability of postoperative refraction, visual acuity, and topographic outcomes, and outcomes based on the safety and efficacy indexes.

Porcine cholecyst–derived scaffold promotes full-thickness wound healing in rabbit

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Deepa Revi

2013-12-01

Full Text Available Graft-assisted healing is an important strategy for treating full-thickness skin wounds. This study evaluated the properties of porcine cholecyst–derived scaffold and its use for treating full-thickness skin wound in rabbit. The physical properties of cholecyst-derived scaffold were congenial for skin-graft application. Compared to a commercially available skin-graft substitute made of porcine small intestinal submucosa, the cholecyst-derived scaffold was rich in natural biomolecules like elastin and glycosaminoglycans. When used as a xenograft, it promoted healing with excess cell proliferation at early phases and acceptable collagen deposition in the later remodelling phases.

Aquaporin expression in the fetal porcine urinary tract changes during gestation

DEFF Research Database (Denmark)

Jakobsen, L K; Trelborg, K; Kingo, P S

2018-01-01

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive....... Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide...

Establishment of a nanoparticle-assisted RT-PCR assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus.

Science.gov (United States)

Zhu, Yu; Wang, Gui-Hua; Cui, Yu-Dong; Cui, Shang-Jin

2016-09-01

Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection.

Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

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Borie Dominic C

2006-03-01

Full Text Available Abstract Background The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3 galactose (αGal present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s with bioartficial liver devices (BALs, composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. Results Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has

Prevention of primary vascular graft infection with silver-coated polyester graft in a porcine model

DEFF Research Database (Denmark)

Gao, H; Sandermann, J; Prag, J

2010-01-01

To evaluate the efficacy of a silver-coated vascular polyester graft in the prevention of graft infection after inoculation with Staphylococcus aureus in a porcine model.......To evaluate the efficacy of a silver-coated vascular polyester graft in the prevention of graft infection after inoculation with Staphylococcus aureus in a porcine model....

Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

OpenAIRE

MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

2013-01-01

Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

Investigation of SNPs in the porcine desmoglein 1 gene

DEFF Research Database (Denmark)

Daugaard, L.; Andresen, Lars Ole; Fredholm, M.

2007-01-01

epidermitis were diagnosed clinically as affected or unaffected. Two regions of the desmoglein I gene were sequenced and genotypes of the SNPs were established. Seven SNPs (823T>C, 828A>G, 829A>G, 830A>T, 831A>T, 838A>C and 1139C>T) were found in the analysed sequences and the allele frequencies were...... the location of single nucleotide polymorphisms (SNPs) in the porcine desmoglein I gene (PIG)DSGI in correlation to the cleavage site as well as if the genotype of the SNPs is correlated to susceptibility or resistance to the disease. Results: DNA from 32 affected and 32 unaffected piglets with exudative...... the genotypes of two out of seven SNPs found in the porcine desmoglein I gene and the susceptibility to exudative epidermitis....

Release of galanin from isolated perfused porcine adrenal glands

DEFF Research Database (Denmark)

Holst, J J; Ehrhart-Bornstein, M; Messell, T

1991-01-01

We found a high concentration of galanin in extracts of porcine adrenal glands (114 pmol/g). By immunohistochemistry, galanin was localized to groups of medullary cells previously shown to produce norepinephrine. To study mechanisms for the release of galanin, we developed the following in vitro...... model: isolated perfused porcine adrenals with intact splanchnic nerve supply. When the nerves were electrically stimulated, epinephrine and norepinephrine secretion increased 276- and 291-fold, respectively, and galanin release increased up to 1,300-fold. Acetylcholine at 10(-6) M stimulated galanin...... release, and hexamethonium almost abolished the response to nerve stimulation. Galanin infusions had no effect on epinephrine and norepinephrine secretion in concentrations of 10(-8) and 10(-7) M, but increased both cortisol and aldosterone secretion (P less than 0.05). Splanchnic nerve stimulation...

A novel porcine cell culture based protocol for the propagation of hepatitis E virus

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Walter Chingwaru

2016-08-01

Full Text Available Objective: To present a comprehensive protocol for the processing of hepatitis E virus (HEV infected samples and propagation of the virus in primary cell cultures. Methods: Hepatitis E was extracted from porcine liver and faecal samples following standard protocols. The virus was then allowed to attach in the presence of trypsin to primary cells that included porcine and bovine intestinal epithelial cells and macrophages over a period of up to 3 h. The virus was propagated by rotational passaging through the cell cultures. Propagation was confirmed by immunoblotting. Results: We developed a comprehensive protocol to propagate HEV in porcine cell model that includes (i rotational culturing of the virus between porcine cell types, (ii pre-incubation of infected cells for 210 min, (iii use of a semi-complete cell culture medium supplemented with trypsin (0.33 µg/mL and (iv the use of simple immunoblot technique to detect the amplified virus based on the open reading frame 2/3. Conclusions: This protocol opens doors towards systematic analysis of the mechanisms that underlie the pathogenesis of HEV in vitro. Using our protocol, one can complete the propagation process within 6 to 9 d.

Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues.

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Tohno, Masanori; Shimosato, Takeshi; Aso, Hisashi; Kitazawa, Haruki

2011-12-15

We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT. Copyright © 2011 Elsevier B.V. All rights reserved.

Experimental porcine cysticercosis using infected beetles with Taenia solium eggs.

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Gomez-Puerta, Luis A; Garcia, Hector H; Gonzalez, Armando E

2018-07-01

Beetles are intermediate hosts for human and animal parasites, and several beetle species have been shown to carry Taenia eggs. An experimental porcine cysticercosis infection model was developed using beetles (Ammophorus rubripes) infected with Taenia solium eggs and then using these beetles for oral pig challenge. A total of 18 three months-old Landrace pigs were divided in four groups. Pigs from groups 1, 2, and 3 (n = 6 pigs per group) were challenged with one, three, and six beetles infected with T. solium eggs, containing approximately 52, 156 or 312 eggs respectively. Pigs were necropsied 12 weeks after infection to assess the presence of T. solium metacestode. Porcine cysticercosis by T. solium was produced in 17 out of 18 pigs (94.4%) challenged with infected beetles, all infected pigs had viable cysts. Only one pig from group 1 was negative to the presence of cysts. The median number of metacestodes per pig in groups 1, 2, and 3 were 2 (range 0-71), 26 (range 5-33) and 40 cysts (range 4-111), respectively. Experimental porcine cysticercosis infection is consistently obtained using beetles as mechanical vectors for T. solium eggs. Copyright © 2018 Elsevier B.V. All rights reserved.

Behaviour of glutathione in the cornea epithelium of cattle and rabbits under the influence of infrared radiation

International Nuclear Information System (INIS)

Scharsich, G.

1978-01-01

After creating a keratitis photoelectrica by means of a mercury/quartz analysis lamp, the concentration of GSH (glutathion, reduced) and GSSG (glutathion, oxidised) in the cornea epithelium of cattle and of rabbits at different times after irradiation was determined. The question of whether ultraviolet radiation considerably influences the GSH/GSSG quotient before the keratitis photoelectrica is fully formed morphologically, could be answered as follows: There is no considerable change in the GSH/GSSG quotient during the first 7-10 hours after the irradiation. Then, when there is, also morphologically, a keratitis photoelectrica, the GSH/GSSG quotient is doubled. The linkage of the GSH/GSSG system and hexosephosphatshunt via the NADP/NADPH system leads us to the conclusion that irradiation does not cause any direct changes in the GSH/GSSG system. Only when the cornea epithelium visably disintegrates an increased reduction of the glutathion occurs, like in several other damaging influences. Thus the found increase of the GSH/GSSG quotient 7-10 hours after ultraviolet irradiation might be unspecific. (orig./AJ) [de

Seroprevalence of Antibodies to Main Porcine Infectious Pathogens in Wild Boars in Some Regions of Russia

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BABORENKO, Elena

2009-01-01

Full Text Available Results of testing 107 serum samples from wild boars (Sus scrofa L., 1758 for thepresence of antibodies to six economically significant porcine infectious disease agents (porcinereproductive and respiratory syndrome (PRRS virus, porcine parvovirus (PPV, swine influenza virus(SIV of H1N1 and H3N2 subtypes, Aujeszky’s disease virus (ADV, porcine transmissiblegastroenteritis virus (TGEV and Mycoplasma hyopneumoniae are presented in the paper. Wild boarwere sampled in seven regions of Russia for diagnostic purposes. The obtained results showed thepresence of antibodies to ADV in 32.5% of samples (83/27, to PPV – in 62% of samples (92/57, toMycoplasma hyopneumoniae – in 52% of samples (98/51. All samples were seronegative to PRRSvirus (107/0, TGEV (91/0 and SIV of H1N1 (89/0 and H3N2 (58/0 subtypes. The researchesdemonstrated the extensive circulation of porcine parvovirus, Aujeszky’s disease virus andMycoplasma hyopneumoniae among Wild boar in some regions of Russia.

No evidence of enteric viral involvement in the new neonatal porcine diarrhoea syndrome in Danish pigs

DEFF Research Database (Denmark)

Goecke, Nicole Bakkegård; Hjulsager, Charlotte Kristiane; Kongsted, H.

2017-01-01

.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected...... with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals...... examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each. Our detailed analyses of piglets from...

The effects of oral and topical corticosteroid in rabbit corneas.

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Araki-Sasaki, Kaoru; Katsuta, Osamu; Mano, Hidetoshi; Nagano, Takashi; Nakamura, Masatsugu

2016-09-05

To determine the most effective route of administration of corticosteroids in the treatment of ocular surface disease, by characterizing the difference between oral prednisolone and topical dexamethasone administration using an animal model. Pharmacokinetic analyses determined the corticosteroid concentrations in the normal ocular tissues of rabbits after oral or topical administration of corticosteroids using LC-MS/MS. In wound healing analyses, the area of the epithelial defect created by keratectomy using a 6-mm trephine was calculated with an image analyzer using an orally or topically steroid-administrated animal model. The average size of basal epithelial cells, the frequency of mitotic basal epithelial cells, the number of squamous cells, and the number of hypertrophic stromal fibroblasts were determined in the enucleated corneal tissues after wound closure. By slit lamp examination, no remarkable differences were observed between orally and topically administered groups. Pharmacokinetic analyses showed that the distribution of dexamethasone after topical administration was superior to that after oral administration in the cornea. In contrast, both concentrations of corticosteroid applied topically and orally were similar with regards to AUCs (area under the concentration-time curve) in the conjunctiva. Although the healing rate was slower in the topical group, all corneas were almost healed within 96 h in the wound healing analysis. According to the histological analyses of epithelial cells, the average basal cell size was larger, the frequency of mitotic basal cells was greater, and the number of squamous epithelial cell layers was lower in the topically administered group although all of these differences were with no statistical significance. However, the number of hypertrophic stromal fibroblasts in the topically administered group was significantly lower than that in the orally administered group. There are different distributions and effects between

Social influence of a religious hero: the late Cardinal Stephen Kim Sou-hwan's effect on cornea donation and volunteerism.

Science.gov (United States)

Bae, Hyuhn-Suhck; Brown, William J; Kang, Seok

2011-01-01

This study examined the mediated influence of a celebrated religious hero in South Korea, Cardinal Stephen Kim, through two forms of involvement--parasocial interaction and identification--on intention toward cornea donation and volunteerism, and it investigated how the news media diffused of his death. A structural equation modeling analysis with a Web-based voluntary survey of more than 1,200 people in South Korea revealed a multistep social influence process, beginning with parasocial interaction with Cardinal Kim, leading to identification with him, which predicted intention toward cornea donation and volunteerism. Additional investigations found that news of Cardinal Kim's death diffused rapidly through media and interpersonal communication. Results of this study demonstrate that religious leaders who achieve a celebrity hero status can prompt public discussion of important issues rather quickly through extensive media coverage, enabling them to promote prosocial behavior and positively affect public health.

Developmental features of porcine haemal nodes: a histological ...

African Journals Online (AJOL)

The result demonstrated progressive changes in the structure of porcine haemal nodes. The capsule and trabeculae of piglet haemal nodes exhibited dense irregular connective tissues with reticular cells and smooth muscle cells. The cortex was more central while the medulla was peripheral with poorly defined boundaries ...

Porcine dermis compared with polypropylene mesh for laparoscopic sacrocolpopexy: a randomized controlled trial.

Science.gov (United States)

Culligan, Patrick J; Salamon, Charbel; Priestley, Jennifer L; Shariati, Amir

2013-01-01

To compare the surgical outcomes 12 months after laparoscopic sacrocolpopexy performed with porcine dermis and the current gold standard of polypropylene mesh. Patients scheduled for laparoscopic sacrocolpopexy were eligible for this randomized controlled trial. Both our clinical research nurse and the patients were blinded as to which material was used. Our primary end point was objective anatomic cure defined as no pelvic organ prolapse quantification (POP-Q) points Stage 2 or greater at any postoperative interval. Our sample size calculation called for 57 patients in each group to achieve 90% power to detect a 23% difference in objective anatomic cure at 12 months (α=0.05). Our secondary end point was clinical cure. Any patient with a POP-Q point greater than zero, or Point C less than or equal to -5, or any complaints of prolapse symptoms whatsoever on Pelvic Floor Distress Inventory-20 or Pelvic Floor Impact Questionnaire, Short Form 7, or reoperation for prolapse were considered "clinical failures"; the rest were "clinical cures." Statistical comparisons were performed using the χ or independent samples t test as appropriate. As expected, there were no preoperative differences between the porcine (n=57) and mesh (n=58) groups. The 12-month objective anatomic cure rates for the porcine and mesh groups were 80.7% and 86.2%, respectively (P=.24), and the "clinical cure" rates for the porcine and mesh groups were 84.2% and 89.7%, respectively (P=.96). Pelvic Floor Distress Inventory-20 and Pelvic Floor Impact Questionnaire, Short Form 7 score improvements were significant for both groups with no differences found between groups. There were no major operative complications. There were similar outcomes in subjective or objective results 12 months after laparoscopic sacrocolpopexy performed with either porcine dermis or polypropylene mesh. ClinicalTrials.gov, www.clinicaltrials.gov, NCT00564083. I.

A simple and rapid identification method for newly emerged porcine Deltacoronavirus with loop-mediated isothermal amplification

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Fanfan Zhang

2017-10-01

Full Text Available Abstract Background Porcine Deltacoronavirus (PDCoV is a newly emerged enteropathogenic coronavirus that causes diarrhea and mortality in neonatal piglets. PDCoV has spread to many countries around the world, leading to significant economic losses in the pork industry. Therefore, a rapid and sensitive method for detection of PDCoV in clinical samples is urgently needed. Results In this study, we developed a single-tube one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay specific for nucleocapsid gene to diagnose and monitor PDCoV infections. The detection limit of RT-LAMP assay was 1 × 101 copies of PDCoV, which was approximately 100-fold more sensitive than gel-based one-step reverse transcription polymerase chain reaction (RT-PCR. This assay could specifically amplify PDCoV and had no cross amplification with porcine epidemic diarrhea virus (PEDV, transmissible gastroenteritis virus (TGEV, porcine kobuvirus (PKoV, porcine astrovirus (PAstV, porcine reproductive and respiratory syndrome virus (PRRSV, classic swine fever virus (CSFV, and porcine circovirus type 2 (PCV2. By screening a panel of clinical specimens (N = 192, this method presented a similar sensitivity with nested RT-PCR and was 1–2 log more sensitive than conventional RT-PCR in detection of PDCoV. Conclusions The RT-LAMP assay established in this study is a potentially valuable tool, especially in low-resource laboratories and filed settings, for a rapid diagnosis, surveillance, and molecular epidemiology investigation of PDCoV infections. To the best of our knowledge, this is the first work for detection of newly emerged PDCoV with LAMP technology.

Microencapsulation of porcine thyroid cell organoids within a polymer microcapsule construct.

Science.gov (United States)

Yang, Yipeng; Opara, Emmanuel C; Liu, Yingbin; Atala, Anthony; Zhao, Weixin

2017-02-01

Hypothyroidism is a common condition of hormone deficiency, and oral administration of thyroid hormones is currently the only available treatment option. However, there are some disadvantages with this treatment modality including compliance challenges to patients. Therefore, a physiologically based alternative therapy for hypothyroidism with little or no side-effects is needed. In this study, we have developed a method for microencapsulating porcine thyroid cells as a thyroid hormone replacement approach. The hybrid wall of the polymer microcapsules permits thyroid hormone release while preventing immunoglobulin antibodies from entry. This strategy could potentially enable implantation of the microcapsule organoids containing allogeneic or xenogeneic thyroid cells to secret hormones over time without the need for immunosuppression of recipients. Porcine thyroid cells were isolated and encapsulated in alginate-poly-L-ornithine-alginate microcapsules using a microfluidic device. The porcine thyroid cells formed three-dimensional follicular spheres in the microcapsules with decent cell viability and proliferation. Thyroxine release from the encapsulated cells was higher than from unencapsulated cells ( P 28 days). These results suggest that the microencapsulated thyroid cell organoids may have the potential to be used for therapy and/or drug screening.

Microscopia confocal de la córnea en facoemulsificación Confocal microscopy of the cornea on phacoemulsification