Microbiologic Examination of Bandage Contact Lenses Used after Corneal Collagen Cross-linking Treatment.

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Yuksel, Erdem; Yalcin, Nuriye Gokçen; Kilic, Gaye; Cubuk, Mehmet Ozgur; Ozmen, Mehmet Cuneyt; Altay, Aylin; Çağlar, Kayhan; Bilgihan, Kamil

2016-01-01

To investigate the agents of bacterial contamination of contact lenses after corneal collagen cross-linking (CCL), and to present the possible changes of ocular flora after riboflavin/ultraviolet A. Seventy-two contact lenses of patients who underwent CCL and 41 contact lenses of patients who underwent photorefractive keratectomy (PRK) as control group were enrolled to the study. After 48 h of incubation, broth culture media was transferred to plates. Samples were accepted as positive if one or more colony-forming units were shown. There were positive cultures in 12 (16.7%) contact lenses in the CCL group and 5 (12.2%) had positive cultures in PRK group. Coagulase-negative staphlycocci (CNS) were the most frequent microorganism. Alpha hemolytic streptococci and Diphteroid spp. were the other isolated microorganisms. Bacterial colonization can occur during and early after the CCL procedure in epithelial healing. To prevent corneal infections after the treatment, prophylactic antibiotics should be prescribed.

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.

Orbscan II anterior elevation changes following corneal collagen cross-linking treatment for keratoconus.

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Tu, Kyaw Lin; Aslanides, Ioannis M

2009-08-01

To analyze anterior corneal elevation changes on Orbscan II following corneal collagen cross-linking (CXL) with riboflavin. This retrospective study included 8 patients (14 eyes) with keratoconus who underwent CXL, with a mean follow-up of 7 months (range: 5 to 10 months). Pre- and postoperative (at last clinic attendance) anterior elevation difference maps were examined for overall patterns of change. On preoperative maps, distances from maximum anterior elevation to pupil center and to topographic geometric center were compared between the two patterns identified. Pre- and postoperative topography, best spectacle-corrected visual acuity (BSCVA), and refraction were also compared between the two patterns. Two patterns of anterior elevation change were visually identified: (1) paracentral steepening, no change, or flattening centrally; and (2) central steepening. The preoperative maps of eyes that manifested pattern 1 had shorter mean distances for maximum anterior elevation to pupil center (1.70 vs. 2.27 mm) and maximum anterior elevation to geometric center (1.45 vs. 1.99 mm) than those that resulted in pattern 2. Mean maximum topographic simulated keratometry decreased (P = .004) and mean irregularity indices at 3 mm (P =.03) and 5 mm (P =.04) were reduced postoperatively in pattern 1 eyes; all increased in pattern 2 eyes. Mean BSCVA improved postoperatively for both patterns. Mean preoperative myopia decreased in pattern 1 eyes by 0.44 diopters (D), whereas it increased for pattern 2 eyes by 1.83 D. Corneal shape change influenced by anisotropy of collagen distribution is a factor in the outcome of CXL treatment for keratoconus.

Protein Oxidation Levels After Different Corneal Collagen Cross-Linking Methods.

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Turkcu, Ummuhani Ozel; Yuksel, Nilay; Novruzlu, Sahin; Yalinbas, Duygu; Bilgihan, Ayse; Bilgihan, Kamil

2016-03-01

To evaluate advanced oxidation protein products (AOPP) levels, superoxide dismutase (SOD) enzyme activity, and total sulfhydryl (TSH) levels in rabbit corneas after different corneal collagen cross-linking (CXL) methods. Eighteen eyes of 9 adult New Zealand rabbits were divided into 3 groups of 6 eyes. The standard CXL group was continuously exposed to UV-A at a power setting of 3 mW/cm for 30 minutes. The accelerated CXL (A-CXL) group was continuously exposed to UV-A at a power setting of 30 mW/cm for 3 minutes. The pulse light-accelerated CXL (PLA-CXL) group received UV-A at a power setting of 30 mW/cm for 6 minutes of pulsed exposure (1 second on, 1 second off). Corneas were obtained after 1 hour of UV-A exposure, and 360-degree keratotomy was performed. SOD enzyme activity, AOPP, and TSH levels were measured in the corneal tissues. Compared with the standard CXL and A-CXL groups (133.2 ± 8.5 and 140.2 ± 6.2 μmol/mg, respectively), AOPP levels were found to be significantly increased in the PLA-CXL group (230.7 ± 30.2 μmol/mg) (P = 0.005 and 0.009, respectively). SOD enzyme activities and TSH levels did not differ between the groups (P = 0.167 and 0.187, respectively). CXL creates covalent bonds between collagen fibers because of reactive oxygen species. This means that more oxygen concentration during the CXL method will produce more reactive oxygen species and, thereby, AOPP. This means that in which CXL method occurs in more oxygen concentration that will produce more reactive oxygen species and thereby AOPP. This study demonstrated that PLA-CXL results in more AOPP formation than did standard CXL and A-CXL.

Herpetic keratitis after corneal collagen cross-linking with riboflavin and ultraviolet-A for progressive keratoconus.

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Yuksel, Nilay; Bilgihan, Kamil; Hondur, Ahmet M

2011-12-01

To describe a case of herpetic keratitis after corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A for progressive keratoconus. A 31-year-old woman with rapidly progressive keratoconus in the left eye was treated with CXL. Four days postoperatively, a dendritic ulcer developed in the treated eye. The diagnosis was confirmed with polymerase chain reaction analysis of the corneal swab for herpes simplex. The patient had no prior history of herpetic eye disease or cold sores. The keratitis resolved in 10 days with treatment. At 1 month, the visual acuity was stable, but a mild superficial opacity was noted. Herpetic keratitis can be induced by CXL even in patients with no history of previous herpetic eye disease. Early diagnosis and proper treatment can facilitate successful management of this rare but important complication.

Riboflavin for corneal cross-linking.

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O'Brart, D P S

2016-06-01

Corneal collagen cross-linking (CXL) with riboflavin and ultraviolet A (UVA) radiation is the first therapeutic modality that appears to arrest the progression of keratoconus and other corneal ectasias. Riboflavin is central to the process, acting as a photosensitizer for the production of oxygen singlets and riboflavin triplets. These free radicals drive the CXL process within the proteins of the corneal stroma, altering its biomechanical properties. Riboflavin also absorbs the majority of the UVA radiation, which is potentially cytotoxic and mutagenic, within the anterior stroma, preventing damage to internal ocular structures, such as the corneal endothelium, lens and retina. Clinical studies report cessation of ectatic progression in over 90% of cases and the majority document significant improvements in visual, keratometric and topographic parameters. Clinical follow-up is limited to 5-10 years, but suggests sustained stability and enhancement in corneal shape. Sight-threatening complications are rare. The optimal stromal riboflavin dosage for CXL is as yet undetermined. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

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Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun; Ren, Li

2015-01-01

Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair

Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid

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Zhao, Xuan; Liu, Yang; Li, Weichang; Long, Kai; Wang, Lin; Liu, Sa; Wang, Yingjun [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou (China)

2015-10-01

Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)–citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col–CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS = 60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30 ± 5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin–eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col–CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications. - Highlights: • Adding different amounts of citric acid could change the properties of films. • The crosslinked films had better mechanical property than non-modified films. • Crosslinked collagen–citric acid films could tolerate suture during operation. • The films showed good ability of epithelial and stromal repair.

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

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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.

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.

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

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Bottos, Katia M.; Oliveira, Anselmo G.; Bersanetti, Patrícia A.; Nogueira, Regina F.; Lima-Filho, Acácio A. S.; Cardillo, José A.; Schor, Paulo; Chamon, Wallace

2013-01-01

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion. PMID:23785497

Simultaneous topography-guided PRK followed by corneal collagen cross-linking after lamellar keratoplasty for keratoconus.

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Spadea, Leopoldo; Paroli, Marino

2012-01-01

The purpose of this paper is to report the results of using combined treatment of customized excimer laser-assisted photorefractive keratectomy (PRK) and prophylactic corneal collagen crosslinking (CXL) for residual refractive error in a group of patients who had previously undergone lamellar keratoplasty for keratoconus. The study included 14 eyes from 14 patients who had originally been treated for keratoconus in one eye by excimer laser-assisted lamellar keratoplasty (ELLK), and subsequently presented with residual ametropia (-6.11 D ± 2.48, range -2.50 to -9.50). After a mean 40.1 ± 12.4 months since ELLK they underwent combined simultaneous corneal regularization treatment with topographically guided transepithelial excimer laser PRK (central corneal regularization) and corneal CXL induced by riboflavin-ultraviolet A. After a mean 15 ± 6.5 (range 6-24) months, all eyes gained at least one Snellen line of uncorrected distance visual acuity (range 1-10). No patient lost lines of corrected distance visual acuity, and four patients gained three lines of corrected distance visual acuity. Mean manifest refractive spherical equivalent was -0.79 ± 2.09 (range +1 to -3.0) D, and topographic keratometric astigmatism was 5.02 ± 2.93 (range 0.8-8.9) D. All the corneas remained clear (haze PRK and corneal CXL provided safe and effective results in the management of corneal regularization for refractive purposes after ELLK for keratoconus.

Treatment Results of Corneal Collagen Cross-Linking Combined with Riboflavin and 440 Nm Blue Light for Bacterial Corneal Ulcer in Rabbits.

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Wei, Shufang; Zhang, Cuiying; Zhang, Shaoru; Xu, Yanyun; Mu, Guoying

2017-10-01

To study the treatment effect of corneal collagen cross-linking (CXL) combined with 440 nm blue light and riboflavin on bacterial corneal ulcer using animal experiments. A total of 21 New Zealand white rabbits that developed Staphylococcus aureus corneal ulcer were randomly divided into three groups. Seven rabbits were used as blank control groups; seven rabbits were treated with CXL combined with riboflavin and 440 nm blue light; and seven rabbits were treated with CXL combined with riboflavin and 370 nm ultraviolet A light. Necrotic tissues or secretions from the ulcer surface, eye secretions, conjunctival hyperemia, hypopyon, corneal infiltration, and pathological changes of the cornea were all observed. The 1st, 3th, and 7th day after CXL treatment, a statistically significant difference was found among the inflammation scores of the three groups. The scores of 440 and 370 groups decreased gradually, significantly lower than that of the control group. Bacterial cultures of 440 and 370 groups turned to be negative while that of the control group remained positive. After 1 day of CXL treatment, pathology pictures of the three groups all showed loss of corneal epithelia with many inflammatory cells in deep stroma. After 7 days of CXL treatment, abscess formed in almost all corneal area in the control group, while in 440 and 370 groups, multilayer healing of corneal epithelia, neovascularization, and many inflammatory cells within ulcers and proliferation of a small amount of fibroblast were seen. CXL combined with riboflavin and 440 nm blue light is effective in treating S. aureus corneal ulcer.

Algorithm for Correcting the Keratometric Error in the Estimation of the Corneal Power in Keratoconus Eyes after Accelerated Corneal Collagen Crosslinking

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David P. Piñero

2017-01-01

Full Text Available Purpose. To analyze the errors associated to corneal power calculation using the keratometric approach in keratoconus eyes after accelerated corneal collagen crosslinking (CXL surgery and to obtain a model for the estimation of an adjusted corneal refractive index nkadj minimizing such errors. Methods. Potential differences (ΔPc among keratometric (Pk and Gaussian corneal power (PcGauss were simulated. Three algorithms based on the use of nkadj for the estimation of an adjusted keratometric corneal power (Pkadj were developed. The agreement between Pk1.3375 (keratometric power using the keratometric index of 1.3375, PcGauss, and Pkadj was evaluated. The validity of the algorithm developed was investigated in 21 keratoconus eyes undergoing accelerated CXL. Results. Pk1.3375 overestimated corneal power between 0.3 and 3.2 D in theoretical simulations and between 0.8 and 2.9 D in the clinical study (ΔPc. Three linear equations were defined for nkadj to be used for different ranges of r1c. In the clinical study, differences between Pkadj and PcGauss did not exceed ±0.8 D nk=1.3375. No statistically significant differences were found between Pkadj and PcGauss (p>0.05 and Pk1.3375 and Pkadj (p<0.001. Conclusions. The use of the keratometric approach in keratoconus eyes after accelerated CXL can lead to significant clinical errors. These errors can be minimized with an adjusted keratometric approach.

A case report of central toxic keratopathy in a patient post TransPRK (followed by corneal collagen cross-linking

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Davey N

2017-04-01

Full Text Available Nicholas Davey, Ioannis M Aslanides, Vasilis Selimis Emmetropia Mediterranean Eye Institute, Heraklion, Crete, Greece Purpose: The purpose of this article is to report a case of central toxic keratopathy in a patient post transepithelial photorefractive keratectomy (TransPRK, followed immediately by corneal collagen cross-linking.Methods: This article describes the case of a 26-year-old male after bilateral aberration-free, TransPRK laser (Schwind Amaris 750S. The procedure was performed for compound myopic astigmatism in November 2015, followed immediately by accelerated corneal collagen cross-linking for early keratoconus.Results: From day 3 post-op, tear film debris underneath both contact lenses with corneal haze and early, progressive central anterior stromal opacity formation only in the left eye were noted. At 2 weeks post-op, the left eye was noted to have a significant hyperopic shift with central corneal thinning in the anterior stroma. A central anterior stromal dense opacity had formed in the left eye with the surrounding superficial stromal haze. As of month 2, the opacity gradually started to improve in size and density. The hyperopic shift peaked at 2 months and continued to improve, largely due to epithelial compensation with a gradual recovery of stromal thickness.Conclusion: The question remains as to what provokes the typical central corneal necrosis/thinning in central toxic keratopathy. We hypothesize that the space between the contact lens and the corneal surface post TransPRK is prone to a “pseudo-interface pathology” that could mimic diffuse lamellar keratitis-like pathology. Suboptimal lid hygiene, resulting in tear film combinations of bacteria, inflammatory cells, matrix metalloproteinases and other proteolytic enzymes, contributes to the degradation of vulnerable, exposed collagen stromal tissue post TransPRK or any surface corneal ablation. Refractive surgeons should maintain a healthy lid margin and tear

Corneal thickness changes during corneal collagen cross-linking with UV-A irradiation and hypo-osmolar riboflavin in thin corneas

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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.

Persistent Epithelial Defects and Corneal Opacity After Collagen Cross-Linking With Substitution of Dextran (T-500) With Dextran Sulfate in Compounded Topical Riboflavin.

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Höllhumer, Roland; Watson, Stephanie; Beckingsale, Peter

2017-03-01

Collagen cross-linking (CXL) is a commonly performed procedure to prevent the progression of keratoconus. Riboflavin is an essential part of the procedure, which facilitates both the cross-linking process and protection of intraocular structures. Dextran can be added to riboflavin to create an isotonic solution. This case report highlights the importance of compounding riboflavin with the correct dextran solution. A retrospective case series. Six eyes of 4 male patients with keratoconus aged from 20 to 38 years underwent CXL with substitution of 20% dextran (T-500) with 20% dextran sulfate in a compounded riboflavin 0.1% solution. Postoperatively, persistent corneal epithelial defects, stromal haze, and then scarring occurred. Corneal transplantation was performed for visual rehabilitation but was complicated by graft rejection followed by failure (n = 1 eye), dehiscence (n = 4), cataract (n = 2), post-laser ablation haze (n = 1), and steroid-induced glaucoma (n = 2). The visual outcome was dextran (T-500) with dextran sulfate in riboflavin solutions during CXL results in loss of vision from permanent corneal opacity. Residual host changes may compromise the results of corneal transplantation.

Thermal and infrared-diode laser effects on indocyanine-green-treated corneal collagen

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Timberlake, George T.; Patmore, Ann; Shallal, Assaad; McHugh, Dominic; Marshall, John

1993-07-01

It has been suggested that laser welds of collagenous tissues form by interdigitation and chemical bonding of thermally 'unraveled' collagen fibrils. We investigated this proposal by attempting to weld highly collagenous, avascular corneal tissue with an infrared (IR) diode laser as follows. First, the temperature at which corneal collagen shrinks and collagen fibrils 'split' into subfibrillary components was determined. Second, since use of a near-IR laser wavelength necessitated addition of an absorbing dye (indocyanine green (ICG) to the cornea, we measured absorption spectra of ICG-treated tissue to ensure that peak ICG absorbance did not change markedly when ICG was present in the cornea. Third, using gel electrophoresis of thermally altered corneal collagen, we searched for covalently crosslinked compounds predicted by the proposed welding mechanism. Finally, we attempted to weld partial thickness corneal incisions infused with ICG. Principal experimental findings were as follows: (1) Human corneal (type I) collagen splits into subfibrillary components at approximately 63 degree(s)C, the same temperature that produces collagen shrinkage. (2) Peak ICG absorption does not change significantly in corneal stroma or with laser heating. (3) No evidence was found for the formation of novel compounds or the loss of proteins as a result of tissue heating. All tissue treated with ICG, however, exhibited a novel 244 kD protein band indicating chemical activity between collagen and corneal stromal components. (4) Laser welding corneal incisions was unsuccessful possibly due to shrinkage of the sides of the incision, lack of incision compression during heating, or a less than optimal combination of ICG concentration and radiant exposure. In summary, these experiments demonstrate the biochemical and morphological complexity of ICG-enhanced IR laser-tissue welding and the need for further investigation of laser welding mechanisms.

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

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Hayes, Sally; Kamma-Lorger, Christina S.; Boote, Craig; Young, Robert D.; Quantock, Andrew J.; Rost, Anika; Khatib, Yasmeen; Harris, Jonathan; Yagi, Naoto; Terrill, Nicholas; Meek, Keith M.

2013-01-01

Purpose To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. Methods One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm2) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm2). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. Results Corneal thickness decreased significantly following riboflavin application (priboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen. PMID:23349690

Corneal collagen cross-linking with riboflavin and ultraviolet - A light for keratoconus: Results in Indian eyes

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Agrawal Vinay

2009-01-01

Full Text Available Aim: To assess the results of corneal collagen cross-linking with riboflavin using ultraviolet - A light for keratoconus at one year in Indian eyes. Materials and Methods: Sixty-eight eyes of 41 patients with progressive keratoconus were included in this retrospective study. All eyes completed was 12 months of follow-up and 37 eyes had a one-year follow-up. The maximum follow-up was 16 months. Ocular examinations including refraction, best corrected visual acuity (BCVA, corneal topography, were recorded at each visit. Results: The mean age was 16.9 ± 3.5 years (range 12-39 years and the mean follow-up was 10.05 ± 3.55 months (range six to 16 months. Thirty seven eyes with a follow-up of at least 12 months were analyzed. The preoperative values on the day of treatment were compared with postoperative values of the 12-month examination. This showed that BCVA improved at least one line in 54% (20/37 of eyes and remained stable in 28% (10/37 of eyes ( P =0.006. Astigmatism decreased by a mean of 1.20 diopter (D in 47% (17/37 of eyes ( P =0.005 and remained stable (within ± 0.50 D in 42% (15/37 of eyes. The K value of the apex decreased by a mean of 2.73 D in 66% (24/37 of eyes ( P =0.004 and remained stable (within ± 0.50 D in 22% (8/37 of eyes. The maximum K value decreased by a mean of 2.47 D in 54% (20/37 of eyes ( P =0.004 and remained stable (within ± 0.50 D in 38% (14/37 of eyes. Corneal Wavefront analysis revealed that spherical and higher-order aberrations did not show significant variations in the follow-up period. The coma component showed a very significant reduction at six months after treatment and persisted throughout the follow-up period ( P =0.003 Conclusion: The results show a stabilization and improvement in keratoconus after collagen cross-linking in Indian eyes. This suggests that it is an effective treatment for progressive keratoconus.

Current status of accelerated corneal cross-linking

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Michael Mrochen

2013-01-01

Full Text Available Corneal cross-linking with riboflavin is a technique to stabilize or reduce corneal ectasia, in diseases such as keratoconus and post-laser-assisted in situ keratomileusis (LASIK ectasia. There is an interest by patient as well as clinicians to reduce the overall treatment time. Especially, the introduction of corneal cross-linking in combination with corneal laser surgery demands a shorter treatment time to assure a sufficient patient flow. The principles and techniques of accelerated corneal cross-linking is discussed.

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

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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.

Conventional Corneal Collagen Cross-Linking Versus Transepithelial Diluted Alcohol and Iontophoresis-Assisted Corneal Cross-Linking in Progressive Keratoconus.

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Bilgihan, Kamil; Yesilirmak, Nilufer; Altay, Yesim; Yuvarlak, Armagan; Ozdemir, Huseyin Baran

2017-12-01

To compare clinical outcomes of conventional corneal cross-linking (C-CXL) and diluted alcohol and iontophoresis-assisted corneal cross-linking (DAI-CXL) for the treatment of progressive keratoconus (KC). Ninety-three eyes of 80 patients with KC were treated by C-CXL (n = 47) or DAI-CXL (n = 46). Visual acuity, keratometry, KC indexes, pachymetry, and aberrations were recorded before treatment and 1, 3, 6, and 12 months after treatment. The demarcation line was assessed 1 month after treatment. A significant improvement in visual acuity was observed at month 3 and month 6 after DAI-CXL and C-CXL, respectively. A significant decrease in maximum keratometry was observed in both groups at month 6. The front symmetry index significantly improved in both groups after 6 months, whereas the Baiocchi Calossi Versaci index significantly improved only after DAI-CXL at month 12 (P = 0.01). Average keratometry and other KC indexes were stable during 12 months of follow-up. Central corneal thickness decreased by 28.6 and 40.2 μm after DAI-CXL and C-CXL at month 1, respectively (P < 0.01), and it reached baseline at the 12th month (P = 0.14) only in the DAI-CXL group. Higher-order aberrations, coma, and spherical aberration significantly worsened at month 1 (P < 0.01) only after C-CXL; however, they improved significantly at month 12 compared with baseline (P < 0.05) in both groups. The demarcation line was visible in all cases at month 1 at a mean depth of 302 ± 56 μm and 311 ± 57 μm after DAI-CXL and C-CXL, respectively (P = 0.7). The DAI-CXL protocol seems as effective as the C-CXL protocol in halting KC progression after 1 year of follow-up.

Novel myopic refractive correction with transepithelial very high-fluence collagen cross-linking applied in a customized pattern: early clinical results of a feasibility study

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Kanellopoulos AJ

2014-04-01

Full Text Available Anastasios John Kanellopoulos LaserVision.gr Institute, Athens, Greece, and New York Medical School, New York, NY, USA Background: The purpose of this study is to report the safety and efficacy of a new application of collagen cross-linking using a novel device to achieve predictable refractive myopic changes in virgin corneas. Methods: Four cases were treated with a novel device employing very high-fluence collagen cross-linking applied in a myopic pattern. Prior to treatment, riboflavin solution was applied to the intact epithelium. The collagen cross-linking device was then engaged for a total of 12 J/cm2, to be applied transepithelially in a predetermined pattern. Cornea clarity, corneal keratometry, and corneal topography were evaluated by both Placido disc and Scheimpflug imaging, along with cornea anterior segment optical coherence tomography and endothelial cell counts. Results: An average of 2.3 diopters was achieved in the first week in all four cases treated with the very high-fluence myopic collagen cross-linking intervention. There was a slight regression to 1.44 diopters at 1 month, which remained stable at 6-month follow-up. The mean keratometry change was from 44.90 diopters to 43.46 diopters. There was no significant change in endothelial cell counts or corneal clarity. There was some mild change in epithelial thickness distribution, with the treated area showing a slight but homogeneous reduction in mean thickness from 52 µm to 44 µm. Conclusion: This report describes the novel application of very high-fluence collagen cross-linking with a predictable well defined myopic refractive (flattening corneal effect. This technique has the advantages of essentially no postoperative morbidity, immediate visual rehabilitation, and the potential for tapering until the desired result is achieved. Keywords: myopia, refractive correction, high-fluence collagen cross-linking, clinical results

Microbiologic, Pharmacokinetic, and Clinical Effects of Corneal Collagen Cross-Linking on Experimentally Induced Pseudomonas Keratitis in Rabbits.

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Cosar, C Banu; Kucuk, Mutlu; Celik, Ekrem; Gonen, Tansu; Akyar, Isin; Serteser, Mustafa; Tokat, Fatma; Ince, Umit

2015-10-01

To determine the effects of corneal collagen cross-linking (CXL) on the penetration of topical 0.5% moxifloxacin, on the number of colony-forming units (CFUs) in the cornea, and on the clinical course in a rabbit eye model of experimentally induced Pseudomonas aeruginosa keratitis. In this prospective animal study, experimental Pseudomonas corneal ulcers were induced in 56 corneas of 28 albino New Zealand rabbits. The corneas were randomly divided into the following 4 groups: the control group (14 eyes), the MOX group (moxifloxacin) (14 eyes), the MOX + CXL group (14 eyes), and the CXL group (14 eyes). On day 4 of the experiment, the eyes in the control group were enucleated and CFU counting was performed. On day 10 of the experiment, all eyes were enucleated and CFU counting was performed. In the MOX and MOX + CXL groups, the moxifloxacin level in the cornea, aqueous humor, iris, plasma, and serum was measured by reverse-phase high-performance liquid chromatography. The difference in the corneal CFU count between the MOX group and the MOX + CXL group was not significant (P = 0.317). Clinical improvement was greatest in the MOX + CXL group (P < 0.001). The mean corneal moxifloxacin level was 0.391 ± 0.09 μg·mg in the MOX group versus 0.291 ± 0.09 μg·mg in the MOX + CXL group; as such, CXL did not have a significant effect on antibiotic penetrance (P = 0.386). Clinical improvement was greatest in the MOX + CXL group. The synergistic effect of CXL on corneal ulcer treatment is not through antibiotic penetrance.

Corneal Collagen Crosslinking Combined with Phototherapeutic Keratectomy and Photorefractive Keratectomy for Corneal Ectasia after Laser in situ Keratomileusis.

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Zhu, Wei; Han, Yunfei; Cui, Changxia; Xu, Wenwen; Wang, Xuan; Dou, Xiaoxiao; Xu, Linlin; Xu, Yanyun; Mu, Guoying

2018-01-01

The aim of this study was to analyze the effects of corneal crosslinking (CXL) combined with phototherapeutic keratectomy (PTK) and photorefractive keratectomy (PRK) in halting the progression and improving the visual function of corneal ectasia after laser in situ keratomileusis (LASIK). PTK-PRK-CXL was performed on 14 eyes of 14 patients who developed corneal ectasia after LASIK. The visual acuity, spherical refraction and cylinder, corneal topography indices, thinnest corneal thickness (TCT), and endothelial cell count were evaluated at baseline and at 1, 3, 6, and 12 months postoperatively. The mean uncorrected visual acuity improved significantly from 0.64 ± 0.36 logMAR preoperatively to 0.19 ± 0.12 logMAR at 12 months of follow-up (p 0.05) beyond 6 months after treatment. PTK-PRK-CXL is a promising procedure to halt the progression of post-LASIK keratectasia with significant visual quality improvement. © 2018 S. Karger AG, Basel.

Comparison of Changes in Central Corneal Thickness During Corneal Collagen Cross-Linking, Using Isotonic Riboflavin Solutions With and Without Dextran, in the Treatment of Progressive Keratoconus.

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Zaheer, Naima; Khan, Wajid Ali; Khan, Shama; Khan, M Abdul Moqeet

2018-03-01

To compare intraoperative changes in central corneal thickness (CCT) during corneal cross-linking, using 2 different isotonic riboflavin solutions either with dextran or with hydroxy propyl methylcellulose, in the treatment of progressive keratoconus. In this retrospective study, we analyzed records of corneal thickness measurements, taken during various steps of cross-linking. Cross-linking was performed using either isotonic riboflavin with dextran (group A) or isotonic riboflavin with hydroxy propyl methylcellulose (without dextran) (group B). CCT measurements were recorded before and after epithelial removal, after saturation with respective isotonic riboflavin solution, after use of hypotonic riboflavin in selected cases, and after ultraviolet A (UV-A) application. A mixed-way analysis of variance was conducted on CCT readings within each group and between both groups, and p dextran causes a significant decrease in corneal thickness, whereas dextran-free isotonic riboflavin causes a significant increase in corneal thickness, thus facilitating the procedure.

Management of pediatric keratoconus - Evolving role of corneal collagen cross-linking: An update

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Vardhaman P Kankariya

2013-01-01

Full Text Available Pediatric keratoconus demonstrates several distinctive management issues in comparison with adult keratoconus with respect to under-diagnosis, poor compliance and modifications in treatment patterns. The major concerns comprise of the accelerated progression of the disease in the pediatric age group and management of co-morbidities such as vernal keratoconjuntivitis. Visual impairment in pediatric patients may affect social and educational development and overall negatively impact their quality of life. The treatment algorithm between adults and pediatric keratoconus has been similar; comprising mainly of visual rehabilitation with spectacles, contacts lenses (soft or rigid and keratoplasty (lamellar or penetrating depending on the stage of the disease. There is a paradigm shift in the management of keratoconus, a new treatment modality, corneal collagen crosslinking (CXL, has been utilized in adult keratoconic patients halting the progression of the disease. CXL has been utilized for over a 10 year period and based on the evidence of efficacy and safety in the adult population; this treatment has been recently utilized in management of pediatric keratoconus. This article will present an update about current management of pediatric keratoconus with special focus on CXL in this age group.

Collagen Cross-Linking: Current Status and Future Directions

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Marine Hovakimyan

2012-01-01

Full Text Available Collagen cross-linking (CXL using UVA light and riboflavin (vitamin B2 was introduced as a clinical application to stabilize the cornea by inducing cross-links within and between collagen fibers. CXL has been investigated extensively and has been shown clinically to arrest the progression of keratoconic or post-LASIK ectasia. With its minimal cost, simplicity, and proven positive clinical outcome, CXL can be regarded as a useful approach to reduce the number of penetrating keratoplasties performed. Small case series have also indicated that CXL is beneficial in corneal edema by reducing stromal swelling behavior and in keratitis by inhibiting pathogen growth. Despite these encouraging results, CXL remains a relatively new method that is potentially associated with complications. Aspects such as side effects and recurrence rates have still to be elucidated. In light of the growing interest in CXL, our paper summarizes present knowledge about this promising approach. We have intentionally endeavored to include the more relevant studies from the recent literature to provide an overview of the current status of CXL.

Safety and efficacy of simultaneous corneal collagen cross-linking with topography-guided PRK in managing low-grade keratoconus: 1-year follow-up.

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Tuwairqi, Waleed S; Sinjab, Mazen M

2012-05-01

To evaluate 1-year visual and topographic outcomes and safety and efficacy of corneal collagen cross-linking (CXL) combined with topography-guided photorefractive keratectomy (TG-PRK) to achieve near emmetropia in eyes with low-grade keratoconus. Twenty-two eyes from 15 patients (11 women, 4 men) were included in a prospective, nonrandomized, noncontrolled clinical study. Mean patient age was 26.6±6.07 years (range: 19 to 40 years). Inclusion criteria were low-grade keratoconus with evidence of progression, transparent cornea, corrected distance visual acuity (CDVA) 0.8 (decimal) or better, corneal thickness >440 μm, and maximum keratometry readings (K-max) PRK with CXL. Study parameters were uncorrected distance visual acuity, CDVA, manifest refractive error, manifest and topographic (corneal) astigmatism, patient satisfaction, and efficacy and safety of the treatment. Follow-up was 1 year. After 1 year, statistically significant improvement was noted in all study parameters (PPRK with CXL is an effective and safe treatment with remarkable visual and topographic outcomes in patients with low-grade keratoconus who meet the recommended inclusion criteria. Copyright 2012, SLACK Incorporated.

Management of corneal ectasia after LASIK with combined, same-day, topography-guided partial transepithelial PRK and collagen cross-linking: the athens protocol.

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Kanellopoulos, Anastasios John; Binder, Perry S

2011-05-01

To evaluate a series of patients with corneal ectasia after LASIK that underwent the Athens Protocol: combined topography-guided photorefractive keratectomy (PRK) to reduce or eliminate induced myopia and astigmatism followed by sequential, same-day ultraviolet A (UVA) corneal collagen cross-linking (CXL). Thirty-two consecutive corneal ectasia cases underwent transepithelial PRK (WaveLight ALLEGRETTO) immediately followed by CXL (3 mW/cm(2)) for 30 minutes using 0.1% topical riboflavin sodium phosphate. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction spherical equivalent, keratometry, central ultrasonic pachymetry, corneal tomography (Oculus Pentacam), and endothelial cell counts were analyzed. Mean follow-up was 27 months (range: 6 to 59 months). Twenty-seven of 32 eyes had an improvement in UDVA and CDVA of 20/45 or better (2.25 logMAR) at last follow-up. Four eyes showed some topographic improvement but no improvement in CDVA. One of the treated eyes required a subsequent penetrating keratoplasty. Corneal haze grade 2 was present in 2 eyes. Combined, same-day, topography-guided PRK and CXL appeared to offer tomographic stability, even after long-term follow-up. Only 2 of 32 eyes had corneal ectasia progression after the intervention. Seventeen of 32 eyes appeared to have improvement in UDVA and CDVA with follow-up >1.5 years. This technique may offer an alternative in the management of iatrogenic corneal ectasia. Copyright 2011, SLACK Incorporated.

Improving the mechanical properties of collagen-based membranes using silk fibroin for corneal tissue engineering.

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Long, Kai; Liu, Yang; Li, Weichang; Wang, Lin; Liu, Sa; Wang, Yingjun; Wang, Zhichong; Ren, Li

2015-03-01

Although collagen with outstanding biocompatibility has promising application in corneal tissue engineering, the mechanical properties of collagen-based scaffolds, especially suture retention strength, must be further improved to satisfy the requirements of clinical applications. This article describes a toughness reinforced collagen-based membrane using silk fibroin. The collagen-silk fibroin membranes based on collagen [silk fibroin (w/w) ratios of 100:5, 100:10, and 100:20] were prepared by using silk fibroin and cross-linking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. These membranes were analyzed by scanning electron microscopy and their optical property, and NaCl and tryptophan diffusivity had been tested. The water content was found to be dependent on the content of silk fibroin, and CS10 membrane (loading 10 wt % of silk fibroin) performed the optimal mechanical properties. Also the suture experiments have proved CS10 has high suture retention strength, which can be sutured in rabbit eyes integrally. Moreover, the composite membrane proved good biocompatibility for the proliferation of human corneal epithelial cells in vitro. Lamellar keratoplasty shows that CS10 membrane promoted complete epithelialization in 35 ± 5 days, and their transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization, and keratoconus are not observed. The composite films show potential for use in the field of corneal tissue engineering. © 2014 Wiley Periodicals, Inc.

AN ANALYTICAL STUDY OF EFFICACY OF CORNEAL COLLAGEN CROSSLINKING C3R PROCEDURE IN PROGRESSIVE KERATOCONUS PATIENTS

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Rajasekar K

2017-10-01

Full Text Available BACKGROUND Keratoconus affects a significant number of the general population with conical weakened protruded area from the cornea due to weakening of the corneal stroma by a genetically premeditated preponderance. We see keratoconus as a standalone disease or accompanying other syndrome manifestations in patients. Mainly, the inferotemporal cornea is affected and the conical protrusion causes profound high irregular myopic astigmatism as a refractive error, which is very difficult to correct in progressed advanced stages. Especially in economically productive age group patients, the poor vision becomes very difficult to live with. Corneal collagen crosslinking procedure is a novel tool in the armamentarium of treatment procedures against this malady. MATERIALS AND METHODS This analytical study was conducted at cornea services, Regional Institute of Ophthalmology and Government Ophthalmic Hospital, Chennai, for a period of 14 months. Forty five eyes of forty patients with early progressive keratoconus who presented to cornea services were subjected to riboflavin UVA collagen crosslinking procedures using a standard protocol after getting an informed consent. Further response to treatment were assessed in the follow up period. RESULTS Out of 40 patients in our series, 23 were males and 17 were females. The maximum patients in our series were in the age group between 10 to 25 yrs. Epi-off procedure was done in 31 eyes and epi-on procedure was done in 14 eyes. The patients with pachymetry 400-450 microns underwent epi-on procedure and more than 450 microns underwent epi-off C3R procedure. The K values in our series were between 49D to maximum 63D. The topographic flattening was seen in 52% in epi-on and epi-off procedures. Vision improvement in our series was 57% following epi-on and 65% following epi-off procedures. CONCLUSION C3R is a very promising therapeutic modality that may halt the progression of ectatic process. It is a less invasive mode

Topographic outcomes after corneal collagen crosslinking in progressive keratoconus: 1-year follow-up

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Mauro C. Tiveron Jr

Full Text Available ABSTRACT Purpose: We aimed to report and analyze topographic and refractive outcomes following corneal collagen crosslinking (CXL in patients with progressive keratoconus (KC. Methods: We performed a retrospective, analytical, and observational study of 100 eyes from 74 progressive KC patients who underwent CXL at the Eye Hospital of Paraná. Keratometric values were analyzed preoperatively as well as 3 and 12 months postoperatively. Results: For a total of 100 eyes, 68 belonged to male patients. The mean age of our study population was 19.9 ± 5.61 years. The average visual acuity and topographic parameters overall were stable after 1 year (p<0.05. After 3 months, steepest keratometry reading (K2 and maximum keratometry (Kmax were significantly decreased (p<0.05. Regarding topographic astigmatism (dK, there was no significant difference between the 3-month and 12-month follow-ups. When we made comparisons between genders following CXL, there were no significant differences related to the changes in Kmax, K2, and spectacle-corrected distance visual acuity (SCDVA. Conclusions: CXL promoted stabilization or improvement of keratometric values and visual acuity. We found that keratoconus apex stability may be achieved 3 months after the procedure. There was no significant difference in keratometric and refractive values measured between male and female patients.

Corneal collagen cross-linking in the stabilization of PRK, LASIK, thermal keratoplasty, and orthokeratology.

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Nguyen, Michelle K; Chuck, Roy S

2013-07-01

To describe the use of corneal collagen cross-linking (CXL) and its efficacy in the stabilization of keratorefractive procedures, including PRK, laser in-situ keratomileusis (LASIK), thermal keratoplasty, and orthokeratology. Since its introduction, CXL has quickly gained interest in the treatment of ectactic disorders due to its ability to increase the biomechanical stability of the cornea. In its earliest use, it has shown to be effective in the treatment of both keratoconus and post-LASIK ectasia. More recent studies of CXL in combination with keratorefractive procedures have shown varying degrees of success. CXL with PRK has shown to be effective in slowing or halting the progression of keratoconus, pellucid marginal degeneration, and post-LASIK ectasia, in addition to potentially decreasing or delaying the need for penetrating keratoplasty. Several small case series have also demonstrated improved stability and efficacy of PRK and LASIK when combined with CXL, as well as a potentially decreased risk of postprocedure ectasia. In conjunction with thermokeratoplasty and orthokeratology, CXL has shown improved but only temporary results in the treatment of keratoconus. Future studies are needed to determine the efficacy and long-term stability of CXL in combination with keratorefractive procedures, as well as to address possible complications.

Long-term results of cornea collagen cross-linking with riboflavin for keratoconus

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Vinay Agrawal

2013-01-01

Full Text Available Corneal collagen cross-linking with riboflavin and UVA light (CXL is the only method designed to arrest the progression of keratoconus. Visual improvement generally starts 3 months after treatment. Reduction is coma seen on aberrometry in early postoperative phase is also responsible for the improvement in visual acuity. In the light of currently available data we can thus say that CXL is a safe procedure that is successful in arresting keratoconus.

Effects of genipin corneal crosslinking in rabbit corneas.

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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.

Changes in corneal topography and biomechanical properties after collagen cross linking for keratoconus: 1-year results.

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Sedaghat, Mohammadreza; Bagheri, Mansooreh; Ghavami, Shahri; Bamdad, Shahram

2015-01-01

To evaluate changes in corneal topography and biomechanical properties after collagen cross-linking (CXL) for progressive keratoconus. Collagen cross-linking was performed on 97 eyes. We assessed uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA). Corneal topography indices were evaluated using placido disc topography, scanning slit anterior topography (Orbscan II), and rotating Scheimpflug topography (Pentacam). Specular microscopy and corneal biomechanics were evaluated. A 1-year-follow-up results revealed that UCVA improved from 0.31 to 0.45 and BCVA changed from 0.78 to 0.84 (P < 0.001). The mean of average keratometry value decreased from 49.62 to 47.95 D (P < 0.001). Astigmatism decreased from 4.84 to 4.24 D (P < 0.001). Apex corneal thickness decreased from 458.11 to 444.46 μm. Corneal volume decreased from 56.66 to 55.97 mm(3) (P < 0.001). Posterior best fit sphere increased from 55.50 to 46.03 mm (P = 0.025). Posterior elevation increased from 99.2 to 112.22 μm (P < 0.001). Average progressive index increased from 2.26 to 2.56 (P < 0.001). A nonsignificant decrease was observed in mean endothelial count from 2996 to 2928 cell/mm(2) (P = 0.190). Endothelial coefficient of variation (CV) increased nonsignificantly from 18.26 to 20.29 (P = 0.112). Corneal hysteresis changed from 8.18 to 8.36 (P = 0.552) and corneal resistance factor increased from 6.98 to 7.21 (P = 0.202), so these changes were not significant. Visual acuity and K values improved after CXL. In spite of the nonsignificant increase in endothelial cell count and increase in the CV, CLX seems to be a safe treatment for keratoconus. Further studies with larger sample sizes and longer follow-up periods are recommended.

Laser welding and collagen crosslinks

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Reiser, K.M.; Last, J.A. [California Univ., Davis, CA (United States). Dept. of Medicine; Small, W. IV; Maitland, D.J.; Heredia, N.J.; Da Silva, L.B.; Matthews, D.L. [Lawrence Livermore National Lab., CA (United States)

1997-02-20

Strength and stability of laser-welded tissue may be influenced, in part, by effects of laser exposure on collagen crosslinking. We therefore studied effects of diode laser exposure (805 nm, 1-8 watts, 30 seconds) + indocyanine green dye (ICG) on calf tail tendon collagen crosslinks. Effect of ICG dye alone on crosslink content prior to laser exposure was investigated; unexpectedly, we found that ICG-treated tissue had significantly increased DHLNL and OHP, but not HLNL. Laser exposure after ICG application reduced elevated DHLNL and OHP crosslink content down to their native levels. The monohydroxylated crosslink HLNL was inversely correlated with laser output (p<0.01 by linear regression analysis). DHLNL content was highly correlated with content of its maturational product, OHP, suggesting that precursor-product relations are maintained. We conclude that: (1)ICG alone induces DHLNL and OHP crosslink formation; (2)subsequent laser exposure reduces the ICG-induced crosslinks down to native levels; (3)excessive diode laser exposure destroys normally occurring HLNL crosslinks.

A study comparing standard and transepithelial collagen cross-linking riboflavin solutions: epithelial findings and pain scores.

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Yuksel, Erdem; Novruzlu, Shahin; Ozmen, Mehmet C; Bilgihan, Kamil

2015-06-01

To evaluate epithelial signs and pain after epithelial-on corneal collagen cross-linking (Epi-on CCL) with new transepithelial riboflavin formulation and epithelial-off corneal collagen cross-linking (Epi-off CCL) with standard riboflavin formulation and to compare pain and duration of epithelial healing between both techniques. Thirty-nine eyes of 39 patients undergoing Epi-on CCL and 39 eyes of 39 patients undergoing Epi-off CCL were evaluated. Corneal epithelial signs and durations of corneal epithelial healing and subjective pain scores after the procedures were recorded and compared between 2 groups. Total epithelialization was observed after 2.7 ± 0.7 days in Epi-on CCL and 2.3 ± 0.4 days in Epi-off CCL (P = 0.006). The mean pain score on the first day was 3.1 ± 0.6 in Epi-on CCL and 2.3 ± 0.4 in Epi-off CCL with a significant difference (P = 0.0001). The epithelial damage was observed in both procedures; also, the epithelial healing time was longer in Epi-on CCL and it is of great importance that the patients should have therapeutic contact lenses until the epithelium heals in both procedures. The Epi-off CCL group had less pain scores than the Epi-on CCL group and more pain problems after Epi-on CCL still remains. The patient should be informed about pain, even if the Epi-on CCL procedure was performed.

Evaluation of Biomechanical Changes in Myopia Patients with Unsatisfactory Corneas After Femto Second-Laser In Situ Keratomileusis (FS-LASIK) Concurrent with Accelerated Corneal Collagen Cross-Linking Using Corvis-ST: Two-Year Follow-Up Results.

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Xu, Weiwei; Tao, Ye; Wang, Liqiang; Huang, Yifei

2017-07-27

BACKGROUND Some myopia patients with unsatisfactory corneas consider corneal refractive surgery for different reasons. Accelerated corneal collagen crosslinking (ACXL) is an effective method to enhance the resistance of the cornea. The present investigation was designed to evaluate the changes of biomechanical properties in patients with myopia and thin corneas after femtosecond-laser in situ keratomileusis (FS-LASIK) concurrent with ACXL. MATERIAL AND METHODS A prospective study was designed. A total of 22 eyes of 11 myopia astigmatism patients with unsatisfactory corneas were enrolled. The patients were assigned to femtosecond-laser in situ keratomileusis concurrent with accelerated corneal collagen crosslinking (FS-LASIK-ACXL). The follow-up duration was 24 months. Manifest refraction, uncorrected (UDVA), and corrected distance visual acuity (CDVA), ultra-high-speed camera (Corvis-ST), corneal topography, anterior segment OCT (AS-OCT), Pentacam, and endothelial cell density (ECD) were examined before and after the operation. The corneal biomechanical and refractive data was analyzed using SAS9.3. Data were analyzed through normal distribution test and variance of analysis. The difference was considered as statistically significant when pLASIK-ACXL operation. The values of first applanation length (A1L), the second applanation length (A2L), the first applanation velocity (A1V), the second applanation velocity (A2V), deformation amplitude (DA), highest concavity peak distance (PD), and radius of curvature at the time of highest concavity (HCR) did not show significant difference after the operation. CONCLUSIONS FS-LASIK-ACXL is an effective and safe surgery for improving visual acuity for myopic patients with thin corneas, and it does not increase the risk of iatrogenic keratectasia.

Corneal Collagen Cross-Linking Combined with an Artiflex Iris-Fixated Anterior Chamber Phakic Intraocular Lens Implantation in a Patient with Progressive Keratoconus

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Sharif Hashmani

2017-10-01

Full Text Available We present here the case of a 24-year-old male who experienced progressive keratoconus and vision loss which adversely affected his ability to carry out everyday tasks. This landed him in the Hashmanis Hospital for consultation. He had a preoperative best corrected visual acuity of 6/12. He underwent multiple Oculus Pentacam examinations, which showed progressive keratoconus. Corneal collagen cross-linking (CXL was performed to stabilize his cornea and, subsequently, an Artiflex anterior chamber iris-fixated phakic intraocular lens (ACIF-PIOL was implanted to alleviate his refractive errors. The patient achieved a postoperative uncorrected visual acuity of 6/12. This report shows that CXL combined with ACIF-PIOL can be safe and effective in those with progressive keratoconus.

Collagen crosslinks in chondromalacia of the patella.

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Väätäinen, U; Kiviranta, I; Jaroma, H; Arokosi, J; Tammi, M; Kovanen, V

1998-02-01

The aim of the study was to determine collagen concentration and collagen crosslinks in cartilage samples from chondromalacia of the patella. To study the extracellular matrix alterations associated to chondromalacia, we determined the concentration of collagen (hydroxyproline) and its hydroxylysylpyridinoline and lysylpyridinoline crosslinks from chondromalacia foci of the patellae in 12 patients and 7 controls from apparently normal cadavers. The structure of the collagen network in 8 samples of grades II-IV chondromalacia was examined under polarized light microscopy. The full-thickness cartilage samples taken with a surgical knife from chondromalacia lesions did not show changes in collagen, hydroxylysylpyridinoline and lysylpyridinoline concentration as compared with the controls. Polarized light microscopy showed decreased birefringence in the superficial cartilage of chondromalacia lesions, indicating disorganization or disappearance of collagen fibers in this zone. It is concluded that the collagen network shows gradual disorganization with the severity of chondromalacia lesion of the patella without changes in the concentration or crosslinks of collagen.

Fabrication of homobifunctional crosslinker stabilized collagen for biomedical application

International Nuclear Information System (INIS)

Lakra, Rachita; Kiran, Manikantan Syamala; Sai, Korrapati Purna

2015-01-01

Collagen biopolymer has found widespread application in the field of tissue engineering owing to its excellent tissue compatibility and negligible immunogenicity. Mechanical strength and enzymatic degradation of the collagen necessitates the physical and chemical strength enhancement. One such attempt deals with the understanding of crosslinking behaviour of EGS (ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)) with collagen to improve the physico-chemical properties. The incorporation of a crosslinker during fibril formation enhanced the thermal and mechanical stability of collagen. EGS crosslinked collagen films exhibited higher denaturation temperature (T d ) and the residue left after thermogravimetric analysis was about 16  ±  5.2%. Mechanical properties determined by uniaxial tensile tests showed a threefold increase in tensile strength and Young’s modulus at higher concentration (100 μM). Water uptake capacity reduced up to a moderate extent upon crosslinking which is essential for the transport of nutrients to the cells. Cell viability was found to be 100% upon treatment with 100 μM EGS whereas only 30% viability could be observed with glutaraldehyde. Rheological studies of crosslinked collagen showed an increase in shear stress and shear viscosity at 37 °C. Crosslinking with EGS resulted in the formation of a uniform fibrillar network. Trinitrobenzene sulfonate (TNBS) assay confirmed that EGS crosslinked collagen by forming a covalent interaction with ε-amino acids of collagen. The homobifunctional crosslinker used in this study enhanced the effectiveness of collagen as a biomaterial for biomedical application. (paper)

Collagen crosslinking for ectasia following PRK performed in excimer laser-assisted keratoplasty for keratoconus.

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Spadea, Leopoldo

2012-01-01

To report the results of corneal collagen crosslinking (CXL) in a patient with corneal ectasia developed after excimer laser-assisted lamellar keratoplasty for keratoconus and a secondary photorefractive keratectomy (PRK) for residual refractive error. A 33-year-old woman, who had originally been treated for keratoconus in the right eye by excimer laser-assisted lamellar keratoplasty, subsequently had her residual ametropia treated by topographically guided, transepithelial excimer laser PRK. Five years after PRK, the patient developed corneal ectasia showing concomitant visual changes of best spectacle-corrected visual acuity (BSCVA) reduced to 20/33 with a refraction of -6.00 +6.00 × 30. The minimum corneal thickness at the ectasia apex was 406 µm. A treatment of riboflavin-UVA-induced corneal CXL was performed on the right eye. Two years after the CXL treatment, the right eye improved to 20/20 BSCVA with a refraction of plano +1.00 × 50 while exhibiting a clear lamellar graft. Corneal CXL provided safe and effective management of ectasia developed after excimer laser-assisted lamellar keratoplasty and PRK.

The Evaluation of Corneal Fragility After UVA/Riboflavin Crosslinking.

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Li, Zhiwei; Wang, Yumeng; Xu, Yanyun; Jhanji, Vishal; Zhang, Chunxiao; Mu, Guoying

2017-03-01

To evaluate the fragility of cornea after UVA/riboflavin crosslinking (CXL). Sixty New Zealand rabbits received UVA/riboflavin crosslinking treatment (wavelength 365 nm, irradiance 3.0 mW/cm, and total dose 5.4 J/cm) on right eyes. Animals were sacrificed before and immediately after treatment (day 0), day 1, 3, 7, and 28 after treatment. A 4×10 mm corneal strip for biomechanical evaluation was harvested after sacrifice. The corneal fragility was evaluated by measurement of elongation rate, whereby the elongation rate equals elongation length/baseline length. The Youngs modulus and maximal stress were 1.41±0.51 MPa and 5.56±1.84 MPa before CXL, and increased to 2.31±0.68 MPa (P=0.008) and 9.25±2.74 MPa (P=0.04), respectively, on day 0, then maintained a stable level within a 28 days follow-up. The elongation rate was 62.04±9.34% before CXL and decreased to 48.95%±8.24% (P=0.02) on day 0, then maintained a stable level within a 28 days follow-up. This study showed an increase in the corneal fragility after UVA/riboflavin crosslinking along with an increase in the corneal stiffness. A long-term follow-up should be taken to evaluate the potential deleterious effect of the increasing corneal fragility after UVA/riboflavin crosslinking.

Crosslinking and corneal cryotherapy in acanthamoeba keratitis -- a histological study.

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Hager, Tobias; Hasenfus, A; Stachon, T; Seitz, B; Szentmáry, N

2016-01-01

Acanthamoeba keratitis is rare, but difficult to treat. Penetrating keratoplasty is performed in therapy-resistant cases. Nevertheless, subsequent recurrences occur in 40 % of the cases. In addition to triple-topical therapy (polyhexamid, propamidinisoethionat, neomycin), treatment alternatives are corneal cryotherapy and/or crosslinking (CXL). The aim of our present histological study was to analyze the persistence of acanthamoebatrophozoites and cysts, the persistence of bacteria, and activation of keratocytes in corneas of acanthamoeba keratitis patients following corneal cryotherapy and/or CXL. We analyzed histologically corneal buttons (from penetrating keratoplasties) of nine patients with acanthamoeba keratitis, following corneal cryotherapy (two patients) or a combination of crosslinking and corneal cryotherapy (seven patients), using haematoxilin–eosin, periodic acid Schiff (PAS), Gram and alpha-smooth muscle actin (alpha-SMA) stainings. Acanthamoeba trophozoites persisted in three corneas after cryotherapy and CXL. Cysts persisted in one of two corneas following corneal cryotherapy and in six of seven corneas after a combination of CXL and cryotherapy. One cornea showed positive Gram staining, but there were no alpha-SMA positive keratocytes in any of the corneas. Crosslinking and corneal cryotherapy have only limited impact on killing of acanthamoeba trophozoites, cysts, or bacteria. Corneal cryotherapy and CXL did not stimulate myofibroblastic transformation of keratocytes.

Distribution of Young's modulus in porcine corneas after riboflavin/UVA-induced collagen cross-linking as measured by atomic force microscopy.

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Jan Seifert

Full Text Available Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young's modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219 ± 67 µm. Within this cross-linked zone, the mean Young's modulus declined from 49 ± 18 kPa at the corneal surface to 46 ± 17 kPa, 33 ± 11 kPa, 17 ± 5 kPa, 10 ± 4 kPa and 10 ± 4 kPa at stromal depth intervals of 0-50 µm, 50-100 µm, 100-150 µm, 150-200 µm and 200-250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface, 7.6 (0-50 µm, 5.4 (50-100 µm, 3.0 (100-150 µm, 1.6 (150-200 µm, and 1.5 (200-250 µm, when compared to the Young's modulus of the posterior 100 µm. The mean Young's modulus within the cross-linked zone was 20 ± 8 kPa (2.9-fold stiffening, while it was 11 ± 4 kPa (1.7-fold stiffening for the entire stroma. Both values were significantly distinct from the mean Young's modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young's modulus in soft

The degree of collagen crosslinks in medical collagen membranes determined by water absorption

International Nuclear Information System (INIS)

Braczko, M.; Tederko, A.; Grzybowski, J.

1994-01-01

Collagen membranes were crosslinked by using three agents: glutaraldehyde, hexametylenediisocyanate, and UV irradiation. The increasing concentrations of above chemical agents or longer time of UV exposition resulted in the higher cross-links degree and in the decrease of collagen membranes swelling (measured as water absorption), their elasticity and mechanical resistance. According to American standards, the degree of collagen biomaterial cross-links is determined by measuring of the digestion time by pepsin. However, that method is very time-consuming. In our study, we have that a simple, linear regression between logarithm of digestion time by pepsin exists and it was identical for all three cross-linking agents used. We have concluded that determination of water absorption can be an alternative, simple and fast method for examination of collagen membrane cross-links degree. (author). 16 refs, 7 figs, 1 tab

Safety and efficacy of collagen crosslinking for the treatment of keratoconus.

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Kolli, Sai; Aslanides, Ioannis M

2010-11-01

Keratoconus is a condition that causes corneal ectasia and reduced vision in young adults. A proportion of these patients have progressive disease requiring corneal transplantation. A revolutionary new treatment that is purported to halt progression of keratoconus, known as collagen crosslinking (CXL), has recently been introduced into clinical practice. CXL involves the treatment of the cornea with riboflavin followed by photoactivation with UVA light leading to corneal strengthening. This article reviews the basic science, clinical protocols, safety aspects and clinical results of CXL. The reader will gain a comprehensive understanding of: i) the basic science of CXL; ii) the optimised protocols for clinical use of CXL; iii) the results of all the main clinical trials in the literature; iv) contraindications to treatment and v) full clinical safety profile of CXL. CXL represents a new treatment that uniquely allows the halt of progression of keratoconus, thus preventing visual loss and the need for surgical intervention. Available data suggest that this treatment has high efficacy and is very safe and may represent the future standard treatment for progressive keratoconus.

Safety and Efficacy of Epithelium-On Corneal Collagen Cross-Linking Using a Multifactorial Approach to Achieve Proper Stromal Riboflavin Saturation

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Aleksandar Stojanovic

2012-01-01

Full Text Available Purpose. To evaluate the efficacy and safety of epithelium-on corneal collagen cross-linking (CXL using a multifactorial approach to achieve proper stromal riboflavin saturation. Methods. This non-randomized retrospective study comprised 61 eyes with progressive keratoconus treated with epithelium-on CXL. Chemical epithelial penetration enhancement (benzalkonium chloride-containing local medication and hypotonic riboflavin solution, mechanical disruption of the superficial epithelium, and prolongation of the riboflavin-induction time until verification of stromal saturation were used before the UVA irradiation. Uncorrected and corrected distance visual acuity (UDVA, CDVA, refraction, corneal topography, and aberrometry were evaluated at baseline and at 1, 3, 6, and 12 months postoperative. Results. At 12-month, UDVA and CDVA improved significantly. None of the eyes lost lines of CDVA, while 27.4% of the eyes gained 2 or more lines. Mean spherical equivalent decreased by 0.74 D, and mean cylindrical reduction was 1.15 D. Irregularity index and asymmetry from Scheimpflug-based topography and Max-K at the location of cone from Placido-based topography showed a significant decrease. Higher-order-aberration data demonstrated a slight reduction in odd-order aberrations S 3, 5,7 (=0.04. Postoperative pain without other complications was recorded. Conclusion. Epithelium-on CXL with our novel protocol appeared to be safe and effective in the treatment of progressive keratoconus.

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.

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation.

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Zhang, Yaqing; Zhang, Xiangmei; Xu, Ling; Wei, Shicheng; Zhai, Maolin

2015-01-01

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via (60)Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.

The structural and optical properties of type III human collagen biosynthetic corneal substitutes

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Hayes, Sally; Lewis, Phillip; Islam, M. Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M.

2015-01-01

The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2–9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106

Superficial corneal crosslinking during laser in situ keratomileusis.

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Seiler, Theo G; Fischinger, Isaak; Koller, Tobias; Derhartunian, Viktor; Seiler, Theo

2015-10-01

To determine the safety of superficial corneal crosslinking after laser in situ keratomileusis (LASIK). Institut für Refraktive und Ophthalmo-Chirurgie, Zurich, Switzerland. Prospective study. Eyes with an ectasia risk score of 2 or higher were treated with standard LASIK (90 μm flap) for myopia correction, after which a rapid corneal crosslinking was performed in the interface (riboflavin 0.5% for 2 minutes, 9 mW/cm(2) for 5 minutes) (Group 1). The follow-up was up to 1 year. The prevalence of complications was statistically compared with that in a group of eyes matched regarding age, sex, and attempted refractive correction that were treated with standard LASIK only (Group 2). One month postoperatively, 5 eyes in Group 1 lost 1 line of corrected distance visual acuity (CDVA) compared with 1 eye in Group 2 (P rate of less than 5%. The refractive success was identical in both groups. Early postoperative complications such as erosions (16%), diffuse lamellar keratitis (DLK) stage 1 (38%), and DLK stage 2 (5%) were statistically significantly more frequent after superficial corneal crosslinking, leading to a statistically significantly reduced uncorrected distance visual acuity at 1 month (P interest in any material or method mentioned. Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

ISOCT study of collagen crosslinking of collagen in cancer models (Conference Presentation)

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Spicer, Graham; Young, Scott T.; Yi, Ji; Shea, Lonnie D.; Backman, Vadim

2016-03-01

The role of extracellular matrix modification and signaling in cancer progression is an increasingly recognized avenue for the progression of the disease. Previous study of field effect carcinogenesis with Inverse Spectroscopic Optical Coherence Tomography (ISOCT) has revealed pronounced changes in the nanoscale-sensitive mass fractal dimension D measured from field effect tissue when compared to healthy tissue. However, the origin of this difference in tissue ultrastructure in field effect carcinogenesis has remained poorly understood. Here, we present findings supporting the idea that enzymatic crosslinking of the extracellular matrix is an effect that presents at the earliest stages of carcinogenesis. We use a model of collagen gel with crosslinking induced by lysyl oxidase (LOXL4) to recapitulate the difference in D previously reported from healthy and cancerous tissue biopsies. Furthermore, STORM imaging of this collagen gel model verifies the morphologic effects of enzymatic crosslinking at length scales as small as 40 nm, close to the previously reported lower length scale sensitivity threshold of 35 nm for ISOCT. Analysis of the autocorrelation function from STORM images of collagen gels and subsequent fitting to the Whittle-Matérn correlation function shows a similar effect of LOXL4 on D from collagen measured with ISOCT and STORM. We extend this to mass spectrometric study of tissue to directly measure concentrations of collagen crosslink residues. The validation of ISOCT as a viable tool for non-invasive rapid quantification of collagen ultrastructure lends it to study other physiological phenomena involving ECM restructuring such as atherosclerotic plaque screening or cervical ripening during pregnancy.

Characterizing the collagen stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation.

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Kishen, Anil; Shrestha, Suja; Shrestha, Annie; Cheng, Calvin; Goh, Cynthia

2016-08-01

Antibacterial and chelating properties of chitosan has been widely studied for various dental applications. To characterize the interaction between chitosan-nanoparticles (CSnp) and collagen, and understand their stabilizing effect against collagenase degradation for dentin matrix stabilization. Phase-1: a single Type I collagen-fibril model was used to study the interaction with CSnp along with carbodiimides crosslinking treatment. Degradation of the crosslinked fibrils was studied with bacterial collagenase enzyme and monitored using Fourier Transform Infrared (FTIR) spectroscopy, turbidity measurement (400nm), ninhydrin assay and Atomic Force Microscopy (AFM). Interaction of CSnp with collagenase and Type I collagen, were evaluated using SDS-PAGE, and proteolytic cleavage potential of a synthetic peptide. Phase-2: degradation of dentin collagen crosslinked with/without CSnp was evaluated using FTIR, ninhydrin assay and Scanning Electron Microscopy (SEM). Glutaraldehyde crosslinking was used as a positive control. Both native collagen-fibrils and dentin collagen after crosslinking showed higher resistance to collagenase degradation, as observed in turbidity measurements and FTIR spectra. AFM images showed the interaction of CSnp with single collagen-fibril and crosslinked collagen resisted collagenase degradation up to 54h. The collagen and collagenase both formed complexes with CSnp resulting in thickening of bands and reduction in collagen degradation. CSnp treated collagenase showed significantly reduced cleavage of the fluorescent peptides. Dentin collagen was coated with CSnp following crosslinking with significant increase in resistance to collagenase degradation. Crosslinked CSnp on collagen stabilized and enhanced the resistance of dentin matrix against bacterial collagenase degradation due to non-specific interaction with both collagen and collagenase. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Comparison of Aqueous Humor Nitric Oxide Levels After Different Corneal Collagen Cross-Linking Methods.

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Yuksel, Nilay; Ozel-Turkcu, Ummuhani; Yalinbas, Duygu; Novruzlu, Sahin; Bilgihan, Ayse; Bilgihan, Kamil

2016-12-01

Nitric oxide production can cause either apoptotic or necrotic cell death through oxidative stress. We aimed to investigate the nitrite oxide metabolites (NO x ) and nitrite levels in the aqueous humor of rabbit eyes after different methods of corneal collagen cross-linking (CXL). Twenty-four eyes of 12 adult New Zealand rabbits were used. They were assigned into four groups, each including six eyes. Group 1 (control) consisted of eyes with no treatment. Group 2 received UV-A power setting at 3 mW/cm 2 for 30 minutes of continuous exposure and named as standard CXL group. Group 3 received UV-A power setting at 30 mW/cm 2 for 3 minutes of continuous exposure and named as accelerated CXL (A-CXL) group. Group 4 received UV-A power setting at 30 mW/cm 2 for 6 minutes of pulsed exposure (1 sec on, 1 sec off) and named as pulse-light accelerated CXL (PLA-CXL). Aqueous humors were aspirated from anterior chamber with a 27G needle after 1 hour UV-A exposure. NO x and nitrite levels were measured Results: The nitrite levels in aqueous humor were significantly increased in Group 2 and Group 3 when compared with Group 1 (p = 0.000, p = 0.036, respectively). When treatment modalities were compared with each other, high nitrite level in Group 2 was statistically significant when compared with Group 4 (p = 0.019). NO x levels were higher in Group 2 when compared with Group 1 (p = 0.006). Numerous studies investigated the physiological and pathophysiological roles of NO. NO is considered one of the most important molecule for ocular health. According to NO x level in aqueous humor, it seems that PLA-CXL is the safest method due to the similar results with control group.

Investigation on fibrous collagen modifications during corneal laser welding by second harmonic generation microscopy

Science.gov (United States)

Matteini, Paolo; Ratto, Fulvio; Rossi, Francesca; Cicchi, Riccardo; Stringari, Chiara; Kapsokalyvas, Dimitrios; Pavone, Francesco S.; Pini, Roberto

2009-02-01

The structural modifications in the collagen lattice of corneal stroma induced by near-infrared laser welding were investigated with second-harmonic generation (SHG) imaging. The corneal laser welding procedure is performed by staining the wound edges with a saturated water solution of Indocyanine Green (ICG) followed by irradiation with a 810 nm diode laser operated in continuous (CWLW: continuous wave laser welding) or pulsed (PLW: pulsed laser welding) mode. Both these procedures can provide closure of corneal wounds by inducing different structural modifications in the extracellular matrix. SHG imaging of native corneal stroma revealed collagen bundles composed of many regularly aligned collagen fibrils. After CWLW the regular lamellar arrangement was lost; collagen bundles appeared densely packed with an increasing disordered arrangement toward the welded cut. The weld was characterized by a loss of details; nevertheless, the observation of the second harmonic signal at this site indicated the lack of collagen denaturation. By contrast, PLW mode produced welding spots at the interface between donor and recipient corneal layers, which were characterized by a severe loss of the SHG signal, suggesting the occurrence of a complete collagen denaturation. SHG imaging appeared to be a powerful tool for visualizing the supramolecular morphological modifications in the collagen matrix after laser welding.

Chitosan Cross-linked Reconstituted Amniotic Collagen Membrane ...

Indian Academy of Sciences (India)

First page Back Continue Last page Overview Graphics. Chitosan Cross-linked Reconstituted Amniotic Collagen Membrane – An Excellent Cell Substratum. The KERATINOCYTE proliferation and Differentiation into multiple layers is due to the presence of type - IV collagen in the amnion. Cultured FIBROBLASTS had good ...

Long-term safety and efficacy follow-up of prophylactic higher fluence collagen cross-linking in high myopic laser-assisted in situ keratomileusis

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Kanellopoulos AJ

2012-07-01

Full Text Available Anastasios John KanellopoulosLaservision.gr Institute, Athens, Greece, and New York University Medical School, New York, NY, USABackground: The purpose of this study was to evaluate the safety and efficacy of ultraviolet A irradiation cross-linking on completion for cases of high myopic laser-assisted in situ keratomileusis (LASIK.Methods: Forty-three consecutive LASIK cases treated with femtosecond laser flap and the WaveLight excimer platform were evaluated perioperatively for uncorrected visual acuity, best corrected spectacle visual acuity, refraction, keratometry, topography, total and flap pachymetry, corneal optical coherence tomography, and endothelial cell count. All eyes at the completion of LASIK had cross-linking through the repositioned flap, with higher fluence (10 mW/cm2 ultraviolet light of an average 370 µm wavelength and 10 mW/cm2 fluence applied for 3 minutes following an earlier single instillation of 0.1% riboflavin within the flap interface. Mean follow-up duration was 3.5 (range 1.0–4.5 years.Results: Mean uncorrected visual acuity changed from 0.2 to 1.2, best corrected spectacle visual acuity from 1.1 to 1.2, spherical equivalent from -7.5 diopters (D to -0.2 D, keratometry from 44.5 D to 38 D, flap pachymetry from 105 µm to, total pachymetry from 525 to 405, and endothelial cell count from 2750 to 2800. None of the cases developed signs of ectasia or significant regression during follow-up.Conclusion: Prophylactic collagen cross-linking for high-risk LASIK cases appears to be a safe and effective adjunctive treatment for refractive regression and potential ectasia. This application may be viewed as prophylactic customization of the biomechanical behavior of corneal collagen.Keywords: prophylactic collagen cross-linking, laser-assisted in situ keratomileusis, high-risk, post-LASIK ectasia

Evaluation of corneal symmetry after UV corneal crosslinking for keratoconus

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Mofty H

2017-11-01

Full Text Available Hanan Mofty,1,2 Khaled Alzahrani,2 Fiona Carley,3 Sophie Harper,3 Arun Brahma,3 Leon Au,3 Debbie Morley,3 M Chantal Hillarby2 1Optometry Department, College of Applied Medical Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, 3Manchester Royal Eye Hospital, Manchester, UK Purpose: The purpose of this study was to assess UV corneal crosslinking (CXL treatment outcomes for keratoconus by evaluating the corneal regularity in patients through follow-up using the Oculus Pentacam.Patients and methods: A total of 18 eyes from CXL patients with keratoconus were studied before and after CXL treatment, and six eyes from six patients who were not treated with CXL served as controls. Treated patients had Pentacam images taken before CXL treatment and regularly 3 months post treatment up to the 12th month. Controls were imaged during their first appointment and after 12 months. Symmetry and asphericity were evaluated and correlated with both best-corrected visual acuity (BCVA and maximum K-readings.Results: In the CXL-treated group, there was a significant improvement in the index of symmetrical variation (ISV and keratoconus index (KI at 3 months and in the index of height asymmetry (IHA and minimum radius of curvature (Rmin at 9 months post treatment. On the contrary, the untreated group’s indices showed some significant worsening in ISV, KI, central keratoconus index (CKI, and Rmin. A novel finding in our study was a slight positive shift of anterior asphericity in the 6 mm, 7 mm, and 8 mm 3 months after treatment, which had a correlation with BCVA (R2=0.390, p=0.053 and a strong correlation with maximum K-reading (R2=0.690, p=0.005. However, the untreated group had no significant changes after 1 year.Conclusion: The corneal asymmetrical shape is associated with the spherical aberration alteration

Corneal Resistance to Keratolysis After Collagen Crosslinking With Rose Bengal and Green Light.

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Fadlallah, Ali; Zhu, Hong; Arafat, Samer; Kochevar, Irene; Melki, Samir; Ciolino, Joseph B

2016-12-01

The purpose of this study was to evaluate the resistance to degradation by collagenase A of corneas that have been crosslinked with Rose Bengal and green light (RGX). The ex vivo crosslinking procedure was performed on enucleated rabbit corneas. Corneas were deepithelialized after applying 30% alcohol. Corneas were stained with Rose Bengal (RB, 0.1%) for 2 minutes and then exposed to green light (532 nm) at 0.25 W/cm2 for times to deliver doses of 50, 100, 150, or 200 J/cm2 (n = 5 per group). Five corneas were pretreated with riboflavin solution (0.1% riboflavin) for 15 minutes and irradiated with ultraviolet A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. Five corneas underwent only de-epithelialization and were otherwise untreated. Five corneas were stained with RB without light exposure. The central corneas of each group was removed with a 8.5-mm trephine and incubated at 37°C in 0.3% collagenase A solution. Time to dissolution of each cornea was compared across treatments. Corneas treated with RGX were treated with light fluences of 50, 100, 150, and 200 J/cm2; these corneas dissolved completely at 8.3 ± 1.2, 11.1 ± 1.4, 12.4 ± 1.7, and 15.7 ± 1.8 hours, respectively. Corneas treated by riboflavin and UVA light dissolved at 15.7 ± 1.7 hours, and nontreated corneas dissolved at 6.1 ± 1.3 hours. Corneas treated with only RB (no green light) dissolved at 9.3 ± 1.7 hours. Compared with the untreated corneas, all of the RB groups and the riboflavin-UVA-treated group of corneas degraded statistically significantly slower than untreated corneas (P < 0.05). Crosslinking with RGX increased corneal resistance to digestion by collagenase comparable to that produced by riboflavin and UVA treatment.

Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells

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Lai JY

2014-11-01

provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering.Keywords: amniotic membrane, L-lysine-pretreated concentration, collagen nanofiber stabilization, limbal progenitor cell preservation, corneal epithelial tissue engineering

Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

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Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

2015-06-01

Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

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Rodriguez-Pascual, Fernando; Slatter, David Anthony

2016-11-23

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens.

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

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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-linking

A novel combined polyphenol-aldehyde crosslinking of collagen film-Applications in biomedical materials.

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Liu, Ting; Shi, Lu; Gu, Zhipeng; Dan, Weihua; Dan, Nianhua

2017-08-01

Despite its crucial role in directing cell fate in healthy and diseased tissues, improvements in physical-chemical properties and biocompatibility of type-I collagen are still needed. In this report, we described combined and facile method to modify collagen. The collagen film was first modified by procyanidins solution, in which, then subjected to further crosslinked by dialdehyde alginate, resulting in collagen-procyanidins-dialdehyde alginate film. The properties of the crosslinked collagen films were investigated and the results were discussed. Results from differential scanning calorimetry and thermo gravimetric analysis suggested that the thermal stabilities of the collagen-procyanidins-dialdehyde alginate film were significantly improved. The mechanical properties of collagen-procyanidins-dialdehyde alginate film in terms of elongation at break and tensile strength increased approximately 2-fold and 3-fold, respectively compare to pure collagen film. In addition, the resistance to collagenase degradation of collagen-procyanidins-dialdehyde alginate film was remarkably promoted. The results from methyltetrazolium assay and confocal laser scanning microscopy showed that no cytotoxicity of collagen film was introduced by the combined crosslinking method. Thus, the novel combined by procyanidins-dialdehyde alginate crosslinking method shown in this study provided a non-toxic and efficient crosslinking method that improved various properties of collagen film, which has great potential applications in biomedical materials. Copyright © 2017 Elsevier B.V. All rights reserved.

In vivo biocompatibility of carbodiimide-crosslinked collagen matrices : Effects of crosslink density, heparin immobilization, and bFGF loading

NARCIS (Netherlands)

van Wachem, PB; Plantinga, JA; Wissink, MJB; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J; van Luyn, MJA

2001-01-01

Collagen matrices, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (E) and N-hydroxvsuccinimide (N), were previously developed as a substrate for endothelial cell seeding of small-diameter vascular grafts. In the present study, the biocompatibility of various EN-crosslinked collagen

Influence of Crosslink Density and Stiffness on Mechanical Properties of Type I Collagen Gel

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Shengmao Lin

2015-02-01

Full Text Available The mechanical properties of type I collagen gel vary due to different polymerization parameters. In this work, the role of crosslinks in terms of density and stiffness on the macroscopic behavior of collagen gel were investigated through computational modeling. The collagen fiber network was developed in a representative volume element, which used the inter-fiber spacing to regulate the crosslink density. The obtained tensile behavior of collagen gel was validated against published experimental data. Results suggest that the cross-linked fiber alignment dominated the strain stiffening effect of the collagen gel. In addition, the gel stiffness was enhanced approximately 40 times as the crosslink density doubled. The non-affine deformation was reduced with the increased crosslink density. A positive bilinear correlation between the crosslink density and gel stiffness was obtained. On the other hand, the crosslink stiffness had much less impact on the gel stiffness. This work could enhance our understanding of collagen gel mechanics and shed lights on designing future clinical relevant biomaterials with better control of polymerization parameters.

Riboflavin-induced photo-crosslinking of collagen hydrogel and its application in meniscus tissue engineering.

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Heo, Jiseung; Koh, Rachel H; Shim, Whuisu; Kim, Hwan D; Yim, Hyun-Gu; Hwang, Nathaniel S

2016-04-01

A meniscus tear is a common knee injury, but its regeneration remains a clinical challenge. Recently, collagen-based scaffolds have been applied in meniscus tissue engineering. Despite its prevalence, application of natural collagen scaffold in clinical setting is limited due to its extremely low stiffness and rapid degradation. The purpose of the present study was to increase the mechanical properties and delay degradation rate of a collagen-based scaffold by photo-crosslinking using riboflavin (RF) and UV exposure. RF is a biocompatible vitamin B2 that showed minimal cytotoxicity compared to conventionally utilized photo-initiator. Furthermore, collagen photo-crosslinking with RF improved mechanical properties and delayed enzyme-triggered degradation of collagen scaffolds. RF-induced photo-crosslinked collagen scaffolds encapsulated with fibrochondrocytes resulted in reduced scaffold contraction and enhanced gene expression levels for the collagen II and aggrecan. Additionally, hyaluronic acid (HA) incorporation into photo-crosslinked collagen scaffold showed an increase in its retention. Based on these results, we demonstrate that photo-crosslinked collagen-HA hydrogels can be potentially applied in the scaffold-based meniscus tissue engineering.

Collagen cross linking: Current perspectives

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Srinivas K Rao

2013-01-01

Full Text Available Keratoconus is a common ectatic disorder occurring in more than 1 in 1,000 individuals. The condition typically starts in adolescence and early adulthood. It is a disease with an uncertain cause and its progression is unpredictable, but in extreme cases, vision deteriorates and can require corneal transplant surgery. Corneal collagen cross-linking (CCL with riboflavin (C3R is a recent treatment option that can enhance the rigidity of the cornea and prevent disease progression. Since its inception, the procedure has evolved with newer instrumentation, surgical techniques, and is also now performed for expanded indications other than keratoconus. With increasing experience, newer guidelines regarding optimization of patient selection, the spectrum of complications and their management, and combination procedures are being described. This article in conjunction with the others in this issue, will try and explore the uses of collagen cross-linking (CXL in its current form.

LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis

DEFF Research Database (Denmark)

Cox, Thomas R; Bird, Demelza; Baker, Ann-Marie

2013-01-01

of metastastic tumor cells. We show that LOX-dependent collagen crosslinking is involved in creating a growth-permissive fibrotic microenvironment capable of supporting metastatic growth by enhancing tumor cell persistence and survival. We show that therapeutic targeting of LOX abrogates not only the extent...... to which fibrosis manifests, but also prevents fibrosis-enhanced metastatic colonization. Finally, we show that the LOX-mediated collagen crosslinking directly increases tumor cell proliferation, enhancing metastatic colonization and growth manifesting in vivo as increased metastasis. This is the first...... time that crosslinking of collagen I has been shown to enhance metastatic growth. These findings provide an important link between ECM homeostasis, fibrosis, and cancer with important clinical implications for both the treatment of fibrotic disease and cancer....

Engineering of Corneal Tissue through an Aligned PVA/Collagen Composite Nanofibrous Electrospun Scaffold.

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Wu, Zhengjie; Kong, Bin; Liu, Rui; Sun, Wei; Mi, Shengli

2018-02-24

Corneal diseases are the main reason of vision loss globally. Constructing a corneal equivalent which has a similar strength and transparency with the native cornea, seems to be a feasible way to solve the shortage of donated cornea. Electrospun collagen scaffolds are often fabricated and used as a tissue-engineered cornea, but the main drawback of poor mechanical properties make it unable to meet the requirement for surgery suture, which limits its clinical applications to a large extent. Aligned polyvinyl acetate (PVA)/collagen (PVA-COL) scaffolds were electrospun by mixing collagen and PVA to reinforce the mechanical strength of the collagen electrospun scaffold. Human keratocytes (HKs) and human corneal epithelial cells (HCECs) inoculated on aligned and random PVA-COL electrospun scaffolds adhered and proliferated well, and the aligned nanofibers induced orderly HK growth, indicating that the designed PVA-COL composite nanofibrous electrospun scaffold is suitable for application in tissue-engineered cornea.

Laser-induced corneal cross-linking upon photorefractive ablation with riboflavin

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Kornilovskiy IM

2016-04-01

Full Text Available Igor M Kornilovskiy,1 Elmar M Kasimov,2 Ayten I Sultanova,2 Alexander A Burtsev1 1Department of Eye Diseases, Federal State Budgetary Institution “National Pirogov Medical Surgical Centre”, Ministry of Health, Moscow, Russia; 2Department of Eye Diseases, Zarifa Aliyeva National Ophthalmology Center, Ministry of Health, Baku, Azerbaijan Aim: To estimate the biomechanical effect of the laser-induced cross-linking resulting from photorefractive ablation of the cornea with riboflavin.Methods: Excimer laser ablation studies were performed ex vivo (32 eyes of 16 rabbits by phototherapeutic keratectomy (PTK and in vivo (24 eyes of 12 rabbits by transepithelial photorefractive keratectomy (TransPRK, with and without riboflavin saturation of the stroma. Then, we performed corneal optical coherence tomography on 36 eyes of 18 patients with varying degrees of myopia at different times after the TransPRK was performed with riboflavin saturation of the stroma.Results: Biomechanical testing of corneal samples saturated with riboflavin revealed cross-linking effect accompanied by the increase in tensile strength and maximum strength. PTK showed increase in tensile strength from 5.1±1.4 to 7.2±1.6 MPa (P=0.001, while TransPRK showed increase in tensile strength from 8.8±0.9 to 12.8±1.3 MPa (P=0.0004. Maximum strength increased from 8.7±2.5 to 12.0±2.8 N (P=0.005 in PTK and from 12.8±1.6 to 18.3±1.2 N (P=0.0004 in TransPRK. Clinical optical coherence tomography studies of the biomicroscopic transparent cornea at different times after TransPRK showed increased density in the surface layers of the stroma and membrane-like structure beneath the epithelium.Conclusion: Photorefractive ablation of the preliminary corneal stroma saturation with riboflavin causes the effect of laser-induced cross-linking, which is attended with an increase in corneal tensile strength, maximum strength, increased density in the surface layers of the stroma, and formation of

Primary Outcomes of Accelerated Epithelium-Off Corneal Cross-Linking in Progressive Keratoconus in Children: A 1-Year Prospective Study

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Sherif A. Eissa

2017-01-01

Full Text Available Purpose. To evaluate corneal transparency following accelerated collagen cross-linking (ACXL in pediatric keratoconus. Design. A prospective interventional case series. Methods. This study included 47 eyes (25 patients, aged 9–14 years, with documented progressive keratoconus. After applying 0.1% riboflavin drops, ACXL was performed. Assessment included corrected distance visual acuity (CDVA, uncorrected visual acuity (UCVA, corneal haze, and corneal densitometry in grayscale units (GSU. Result. The mean baseline and corneal densitometry peaked at 3 months post-ACXL while central and posterior densitometry showed a statistically significant increase (P<0.05 and peaked at 8 months postoperatively. By 12 months, densitometry in all corneal layers (P≥0.99 and concentric zones (P≥0.97 reached near baseline values. Slit-lamp graded haze peaked at 1 month to 1.82 ± 0.65 (P<0.05 and declined to near baseline at 12 months (0.39 ± 0.58. There was a statistically significant increase in the mean UCVA and CDVA at 12 months. Conclusion. Total and anterior corneal densitometry peaked after 3 months, while central and posterior densitometry peaked after 8 months. Maximum haze was at 1 month post-ACXL. All corneal layers, concentric zone densitometry and haze reached near baseline values after 1 year. Scheimpflug densitometry showed weak correlation with CDVA over the 12-month follow-up period (r=−0.193.

Glyoxal Crosslinking of Cell-Seeded Chitosan/Collagen Hydrogels for Bone Regeneration

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Wang, Limin; Stegemann, Jan P.

2011-01-01

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation, and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1 mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in 3D matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects. PMID:21345389

Scleral lens tolerance after corneal cross-linking for keratoconus

NARCIS (Netherlands)

Visser, Esther Simone; Soeters, Nienke; Tahzib, Nayyirih G.

2015-01-01

Purpose. Subjective and objective evaluation of scleral lens tolerance and fitting before and after corneal cross-linking (CXL) for progressive keratoconus. Methods. In this prospective cohort, evaluations were made of 18 unilateral eyes in patients who underwent CXL and had been wearing scleral

RELATIONS BETWEEN INVITRO CYTOTOXICITY AND CROSS-LINKED DERMAL SHEEP COLLAGENS

NARCIS (Netherlands)

VANLUYN, MJA; VANWACHEM, PB; DAMINK, LO; DIJKSTRA, PJ; FEIJEN, J; NIEUWENHUIS, P

Collagen-based biomaterials have found various applications in the biomedical field. However, collagen-based biomaterials may induce cytotoxic effects. This study evaluated possible cytotoxic effects of (crosslinked) dermal sheep collagen (DSC) using a 7-d-methylcellulose cell culture with human

Combined application of prophylactic corneal cross-linking and laser in-situ keratomileusis - a review of literature.

Science.gov (United States)

Chan, Tommy C Y; Ng, Alex L K; Chan, Karen K W; Cheng, George P M; Wong, Ian Y H; Jhanji, Vishal

2017-11-01

Laser in-situ keratomileusis (LASIK) is safe and effective laser refractive procedures in treating refractive errors. However, regression of treatment and iatrogenic keratectasia remain to be a major concern, especially in treating thin cornea with high ametropia. Collagen cross-linking (CXL) is an effective method in stopping keratoconus progression through increasing the biomechanical strength of the cornea. Adjuvant cross-linking to refractive procedures can theoretically help prevent regression and reduce the risk of keratectasia development by increasing the mechanical stability of cornea. During the procedure, riboflavin is directly applied to the corneal stroma, thereby reducing the need of de-epithelialization as in the conventional protocol for keratoconus. Currently, there is still no consensus regarding the indication of CXL during refractive procedure, nor any standardized treatment protocol. This article aims to summarize the current evidence regarding the use of adjuvant CXL in LASIK. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

[Riboflavin UVA crosslinking in progressive keratoconus].

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Maier, P; Reinhard, T

2017-06-01

In patients with keratoconus, a progressive, ectatic disease of the cornea, the shape of the cornea is continuously changing leading to a reduction in visual acuity by progressive myopia and more and more (irregular) astigmatism. The symptomatic treatment consists of the prescription of glasses or special gas-permeable rigid contact lenses. Corneal tomography is generally used for diagnosis. After initial diagnosis of keratoconus, regular tomographic follow-ups should be performed. If clinically significant progression is found and confirmed by repeated measurements, riboflavin UVA collagen crosslinking should be offered to the patients. The aim of riboflavin UVA collagen crosslinking is to halt the progression of the disease to avoid further complications. The therapeutic principle is a combined effect of the photosensitizer riboflavin and UVA light. This stiffening effect of the corneal tissue halts the progression of keratoconus. The efficacy of this treatment has been demonstrated in various randomized, controlled trials.

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

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Gábor Sirokmány

2018-06-01

Full Text Available Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels. Keywords: Peroxidasin, NADPH oxidase, Hydrogen peroxide, Collagen IV, Sulfilimine

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation

NARCIS (Netherlands)

Wissink, M.J.B.; Beernink, R.; Pieper, J.S.; Poot, Andreas A.; Engbers, G.H.M.; Beugeling, T.; Beugeling, T.; van Aken, W.G.; Feijen, Jan

2001-01-01

In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free

Endothelial cell seeding on crosslinked collagen : Effects of crosslinking on endothelial cell proliferation and functional parameters

NARCIS (Netherlands)

Wissink, MJB; van Luyn, MJA; Dijk, F; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper

Two-photon induced collagen cross-linking in bioartificial cardiac tissue

Science.gov (United States)

Kuetemeyer, Kai; Kensah, George; Heidrich, Marko; Meyer, Heiko; Martin, Ulrich; Gruh, Ina; Heisterkamp, Alexander

2011-08-01

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

NARCIS (Netherlands)

Damink, LHHO; Dijkstra, PJ; vanLuyn, MJA; vanWachem, PB; Nieuwenhuis, P; Feijen, J

A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). Cross-linking using EDC involves the activation of carboxylic acid groups to give O-acylisourea groups, which form cross-links

Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

NARCIS (Netherlands)

Olde damink, L.H.H.; Olde Damink, L.H.H.; Dijkstra, Pieter J.; van Luyn, M.J.A.; van Wachem, P.B.; Nieuwenhuis, P.; Feijen, Jan

1996-01-01

A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). Cross-linking using EDC involves the activation of carboxylic acid groups to give O-acylisourea groups, which form cross-links

Collagen crosslink location: a molecular marker for fibrosis in lungs of rats with experimental silicosis

International Nuclear Information System (INIS)

Gerriets, J.E.; Reiser, K.M.; Last, J.A.

1986-01-01

Collagen content is increased in lungs of animals with experimental silicosis. They hypothesize that the collagen deposited in such fibrotic lungs differs structurally from normal lung collagen. Silicotic lung collagen shows an increase in lysine hydroxylation. In addition, the ratio of the difunctional crosslinks DHLNL (dihydroxylysinonorleucine) to HLNL (hydroxylysinonorleucine) is sharply elevated compared to that in control lungs. The peptide α1(I)CB7 x α2(I)CB1 crosslinked by HLNL was demonstrated in NaB 3 H 4 -reduced, CNBr-digested collagen from rat tail tendon by peptide purification, followed by periodate oxidation and amino acid analysis. Further structural analysis of this peptide was obtained by digestion of the crosslinked peptide with trypsin and purification of the tryptic peptide containing this crosslink followed by amino acid analysis. They then examined the analogous collagenous peptide in normal and silicotic lungs and analyzed the crosslink it contained. They observed that DHLNL was present at specific sites previously containing HLNL; that is, the collagen in fibrotic lungs is altered at specific sites by post-translational modification of a lysine residue by hydroxylation in a predictable way. They conclude that such unusual hydroxylation of a specific lysine residue in the α2 chain provides a molecular marker for fibrotic lung collagen

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

Science.gov (United States)

Mižíková, Ivana; Ruiz-Camp, Jordi; Steenbock, Heiko; Madurga, Alicia; Vadász, István; Herold, Susanne; Mayer, Konstantin; Seeger, Werner; Brinckmann, Jürgen; Morty, Rory E

2015-06-01

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia. Copyright © 2015 the American Physiological Society.

Studies on Cross-linking of succinic acid with chitosan/collagen

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Tapas Mitra

2013-01-01

Full Text Available The present study summarizes the cross-linking property of succinic acid with chitosan /collagen. In detail, the chemistry behind the cross-linking and the improvement in mechanical and thermal properties of the cross-linked material were discussed with suitable instruments and bioinformatics tools. The concentration of succinic acid with reference to the chosen polymers was optimized. A 3D scaffold prepared using an optimized concentration of succinic acid (0.2% (w/v with chitosan (1.0% (w/v and similarly with collagen (0.5% (w/v, was subjected to surface morphology, FT-IR analysis, tensile strength assessment, thermal stability and biocompatibility. Results revealed, cross-linking with succinic acid impart appreciable mechanical strength to the scaffold material. In silico analysis suggested the prevalence of non-covalent interactions, which played a crucial role in improving the mechanical and thermal properties of the cross-linked scaffold. The resultant 3D scaffold may find application as wound dressing material, as an implant in clinical applications and as a tissue engineering material.

The theory and art of corneal cross-linking.

Science.gov (United States)

McQuaid, Rebecca; Cummings, Arthur B; Mrochen, Michael

2013-08-01

Before the discovery of corneal cross-linking (CXL), patients with keratoconus would have had to undergo corneal transplantation, or wear rigid gas permeable lenses (RGPs) that would temporarily flatten the cone, thereby improving the vision. The RGP contact lens (CL) would not however alter the corneal stability and if the keratoconus was progressive, the continued steepening of the cone would occur under the RGP CL. To date, the Siena Eye has been the largest study to investigate long term effects of standard CXL. Three hundred and sixty-three eyes were treated and monitored over 4 years, producing reliable long-term results proving long-term stability of the cornea by halting the progression of keratoconus, and proving the safety of the procedure. Traditionally, CXL requires epithelial removal prior to corneal soakage of a dextran-based 0.1% riboflavin solution, followed by exposure of ultraviolet-A (UV-A) light for 30 min with an intensity of 3 mW/cm2. A series of in vitro investigations on human and porcine corneas examined the best treatment parameters for standard CXL, such as riboflavin concentration, intensity, wavelength of UV-A light, and duration of treatment. Photochemically, CXL is achieved by the generation of chemical bonds within the corneal stroma through localized photopolymerization, strengthening the cornea whilst minimizing exposure to the surrounding structures of the eye. In vitro studies have shown that CXL has an effect on the biomechanical properties of the cornea, with an increased corneal rigidity of approximately 70%. This is a result of the creation of new chemical bonds within the stroma.

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate

NARCIS (Netherlands)

Pieper, J.S.; Oosterhof, A.; Dijkstra, Pieter J.; Veerkamp, J.H.; van Kuppevelt, T.H.

1999-01-01

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked

Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model.

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Natalia Vázquez

Full Text Available Corneal keratoplasty (penetrating or lamellar using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of

Efficacy of iontophoresis-assisted epithelium-on corneal cross-linking for keratoconus

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Hong-Zhen Jia

2018-04-01

Full Text Available Corneal cross-linking (CXL is a noninvasive therapeutic procedure for keratoconus that is aimed at improving corneal biomechanical properties by induction of covalent cross-links between stromal proteins. It is accomplished by ultraviolet A (UVA radiation of the cornea, which is first saturated with photosensitizing riboflavin. It has been shown that standard epithelium-off CXL (S-CXL is efficacious, and it has been recommended as the standard of care procedure for keratoconus. However, epithelial removal leads to pain, transient vision loss, and a higher risk of corneal infection. To avoid these disadvantages, transepithelial CXL was developed. Recently, iontophoresis has been adopted to increase riboflavin penetration through the epithelium. Several clinical observations have demonstrated the safety and efficacy of iontophoresis-assisted epithelium-on CXL (I-CXL for keratoconus. This review aimed to provide a comprehensive summary of the published studies regarding I-CXL and a comparison between I-CXL and S-CXL. All articles used in this review were mainly retrieved from the PubMed database. Original articles and reviews were selected if they were related to the I-CXL technique or related to the comparison between I-CXL and S-CXL.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

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Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

2016-04-29

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen*

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A.; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M.; Marini, Joan C.; Yamauchi, Mitsuo

2016-01-01

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD.

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Christina S Kamma-Lorger

Full Text Available The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD. In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT in the decorin (DCN gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS, to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

BIOCOMPATIBILITY AND TISSUE REGENERATING CAPACITY OF CROSS-LINKED DERMAL SHEEP COLLAGEN

NARCIS (Netherlands)

VANWACHEM, PB; VANLUYN, MJA; DAMINK, LHHO; DIJKSTRA, PJ; FEIJEN, J; NIEUWENHUIS, P

The biocompatibility and tissue regenerating capacity of four crosslinked dermal sheep collagens (DSC) was studied. In vitro, the four DSC versions were found to be noncytotoxic or very low in cytoxicity. After subcutaneous implantation in rats, hexamethylenediisocyanate-crosslinked DSC (HDSC)

The theory and art of corneal cross-linking

Directory of Open Access Journals (Sweden)

Rebecca McQuaid

2013-01-01

Full Text Available Before the discovery of corneal cross-linking (CXL, patients with keratoconus would have had to undergo corneal transplantation, or wear rigid gas permeable lenses (RGPs that would temporarily flatten the cone, thereby improving the vision. The RGP contact lens (CL would not however alter the corneal stability and if the keratoconus was progressive, the continued steepening of the cone would occur under the RGP CL. To date, the Siena Eye has been the largest study to investigate long term effects of standard CXL. Three hundred and sixty-three eyes were treated and monitored over 4 years, producing reliable long-term results proving long-term stability of the cornea by halting the progression of keratoconus, and proving the safety of the procedure. Traditionally, CXL requires epithelial removal prior to corneal soakage of a dextran-based 0.1% riboflavin solution, followed by exposure of ultraviolet-A (UV-A light for 30 min with an intensity of 3 mW/cm2. A series of in vitro investigations on human and porcine corneas examined the best treatment parameters for standard CXL, such as riboflavin concentration, intensity, wavelength of UV-A light, and duration of treatment. Photochemically, CXL is achieved by the generation of chemical bonds within the corneal stroma through localized photopolymerization, strengthening the cornea whilst minimizing exposure to the surrounding structures of the eye. In vitro studies have shown that CXL has an effect on the biomechanical properties of the cornea, with an increased corneal rigidity of approximately 70%. This is a result of the creation of new chemical bonds within the stroma.

Microscopic characterization of collagen modifications induced by low-temperature diode-laser welding of corneal tissue.

Science.gov (United States)

Matteini, Paolo; Rossi, Francesca; Menabuoni, Luca; Pini, Roberto

2007-08-01

Laser welding of corneal tissue that employs diode lasers (810 nm) at low power densities (12-20 W/cm(2)) in association with Indocyanine Green staining of the wound is a technique proposed as an alternative to conventional suturing procedures. The aim of this study is to evaluate, by means of light (LM) and transmission electron microscopy (TEM) analyses, the structural modifications induced in laser-welded corneal stroma. Experiments were carried out in 20 freshly enucleated pig eyes. A 3.5 mm in length full-thickness cut was produced in the cornea, and was then closed by laser welding. Birefringence modifications in samples stained with picrosirius red dye were analyzed by polarized LM to assess heat damage. TEM analysis was performed on ultra-thin slices, contrasted with uranyl acetate and lead citrate, in order to assess organization and size of type I collagen fibrils after laser welding. LM evidenced bridges of collagen bundles between the wound edges, with a loss of regular lamellar organization at the welded site. Polarized LM indicated that birefringence properties were mostly preserved after laser treatment. TEM examinations revealed the presence of quasi-ordered groups of fibrils across the wound edges preserving their interfibrillar spacing. These fibrils appeared morphologically comparable to those in the control tissue, indicating that type I collagen was not denatured during the diode laser corneal welding. The preservation of substantially intact, undenatured collagen fibrils in laser-welded corneal wounds supported the thermodynamic studies that we carried out recently, which indicated temperatures below 66 degrees C at the weld site under laser irradiation. This observation enabled us to hypothesize that the mechanism, proposed in the literature, of unwinding of collagen triple helixes followed by fibrils "interdigitation" is not likely to occur in the welding process that we set up for the corneal suturing.

Pulsed Light Accelerated Crosslinking versus Continuous Light Accelerated Crosslinking: One-Year Results

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Cosimo Mazzotta

2014-01-01

Full Text Available Purpose. To compare functional results in two cohorts of patients undergoing epithelium-off pulsed (pl-ACXL and continuous light accelerated corneal collagen crosslinking (cl-ACXL with dextran-free riboflavin solution and high-fluence ultraviolet A irradiation. Design. It is a prospective, comparative, and interventional clinical study. Methods. 20 patients affected by progressive keratoconus were enrolled in the study. 10 eyes of 10 patients underwent an epithelium-off pl-ACXL by the KXL UV-A source (Avedro Inc., Waltham, MS, USA with 8 minutes (1 sec. on/1 sec. off of UV-A exposure at 30 mW/cm2 and energy dose of 7.2 J/cm2; 10 eyes of 10 patients underwent an epithelium-off cl-ACXL at 30 mW/cm2 for 4 minutes. Riboflavin 0.1% dextran-free solution was used for a 10-minutes corneal soaking. Patients underwent clinical examination of uncorrected distance visual acuity and corrected distance visual acuity (UDVA and CDVA, corneal topography and aberrometry (CSO EyeTop, Florence, Italy, corneal OCT optical pachymetry (Cirrus OCT, Zeiss Meditec, Jena, Germany, endothelial cells count (I-Conan Non Co Robot, and in vivo scanning laser confocal microscopy (Heidelberg, Germany at 1, 3, 6, and 12 months of follow-up. Results. Functional results one year after cl-ACXL and pl-ACXL demonstrated keratoconus stability in both groups. Functional outcomes were found to be better in epithelium-off pulsed light accelerated treatment together with showing a deeper stromal penetration. No endothelial damage was recorded during the follow-up in both groups. Conclusions. The study confirmed that oxygen represents the main driver of collagen crosslinking reaction. Pulsed light treatment optimized intraoperative oxygen availability improving postoperative functional outcomes compared with continuous light treatment.

Radiation crosslinking of poly(butyl acrylate) during polymerization and grafted copolymerization with Cr(III) crosslinked collagen

International Nuclear Information System (INIS)

Pietrucha, K.; Kroh, J.

1984-01-01

Enhanced crosslinking of synthetic polymer simultaneous with grafting and homopolymerization processes have been observed in irradiated leather tanned with Cr(III) and embedded with aqueous emulsions of butyl acrylate. Extent of poly(butyl acrylate) crosslinking during copolymerization was found to be approximately one order higher than in the case of radiation polymerization of butyl acrylate in emulsion. New method for isolation of grafted copolymer based on degradation of collagen has been developed. The extent of crosslinking was calculated from the swelling data. (author)

The mechanism of collagen cross-linking in diabetes: a puzzle nearing resolution.

Science.gov (United States)

Monnier, V M; Glomb, M; Elgawish, A; Sell, D R

1996-07-01

Considerable interest has been focused in recent years on the mechanism of collagen cross-linking by high glucose in vitro and in vivo. Experiments in both diabetic humans and in animals have shown that over time collagen becomes less soluble, less digestible by collagenase, more stable to heat-induced denaturation, and more glycated. In addition, collagen becomes more modified by advanced products of the Maillard reaction, i.e., immunoreactive advanced glycation end products and the glycoxidation markers carboxymethyllysine and pentosidine. Mechanistic studies have shown that collagen cross-linking in vitro can be uncoupled from glycation by the use of antioxidants and chelating agents. Experiments in the authors' laboratory revealed that approximately 50% of carboxymethyllysine formed in vitro originates from pathways other than oxidation of Amadori products, i.e., most likely the oxidation of Schiff base-linked glucose. In addition, the increase in thermal stability of rat tail tendons exposed to high glucose in vitro or in vivo was found to strongly depend on H2O2 formation. The final missing piece of the puzzle is that of the structure of the major cross-link. We speculate that it is a nonfluorescent nonultraviolet active cross-link between two lysine residues, which includes a fragmentation product of glucose linked in a nonreducible bond labile to both strong acids and bases.

A novel collagen film with micro-rough surface structure for corneal epithelial repair fabricated by freeze drying technique

International Nuclear Information System (INIS)

Liu, Yang; Ren, Li; Wang, Yingjun

2014-01-01

Highlights: • Collagen film with micro-rough surface is fabricated by freeze drying technique. • The film has suitable water uptake capability and toughness performance. • The film has good optical performance. • Human corneal epithelial cells studies confirmed the biocompatibility of the film. - Abstract: Corneal epithelial defect is a common disease and keratoplasty is a common treatment method. A collagen film with micro-rough surface was fabricated through a simple freeze drying technique in this study. Compared with the air-dried collagen film (AD-Col), this freeze-dried collagen film (FD-Col) has a more suitable water uptake capability (about 85.5%) and toughness performance. Both of the two films have good optical properties and the luminousness of them is higher than 80%. Besides, the adhesion and proliferation rate of human corneal epithelial cells on the micro-rough surface of FD-Col film is higher than that on the smooth surface of AD-Col film. The results indicate that this FD-Col film may have potential applications for corneal epithelial repair

A novel collagen film with micro-rough surface structure for corneal epithelial repair fabricated by freeze drying technique

Energy Technology Data Exchange (ETDEWEB)

Liu, Yang [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Ren, Li, E-mail: psliren@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wang, Yingjun, E-mail: imwangyj@163.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China)

2014-05-01

Highlights: • Collagen film with micro-rough surface is fabricated by freeze drying technique. • The film has suitable water uptake capability and toughness performance. • The film has good optical performance. • Human corneal epithelial cells studies confirmed the biocompatibility of the film. - Abstract: Corneal epithelial defect is a common disease and keratoplasty is a common treatment method. A collagen film with micro-rough surface was fabricated through a simple freeze drying technique in this study. Compared with the air-dried collagen film (AD-Col), this freeze-dried collagen film (FD-Col) has a more suitable water uptake capability (about 85.5%) and toughness performance. Both of the two films have good optical properties and the luminousness of them is higher than 80%. Besides, the adhesion and proliferation rate of human corneal epithelial cells on the micro-rough surface of FD-Col film is higher than that on the smooth surface of AD-Col film. The results indicate that this FD-Col film may have potential applications for corneal epithelial repair.

Late onset of a persistent, deep stromal scarring after PRK and corneal cross-linking in a patient with forme fruste keratoconus.

Science.gov (United States)

Güell, Jose L; Verdaguer, Paula; Elies, Daniel; Gris, Oscar; Manero, Felicidad

2014-04-01

To present a case of a late, deep stromal scar in a 22-year-old patient with forme fruste keratoconus who underwent combined corneal cross-linking and photorefractive keratectomy (PRK). Topography-guided corneal cross-linking combined with corneal PRK (without complications) was performed in both eyes with a delay of 2 weeks between each eye. At the 5-month postoperative examination of the right eye, a localized corneal haze was circumscribed to the posterior deep stroma, signifying a decrease of visual acuity. However, this improved partially and temporarily when treated with topical corticoids during 2 years of follow-up and then reoccurred, affecting the corrected distance visual acuity. To the authors' knowledge, this is the first documented, clinical case presenting a deep stromal affectation without endothelial decompensation and visual acuity affectation as a postoperative complication following topography-guided PRK and corneal cross-linking. Copyright 2014, SLACK Incorporated.

Intraoperative Optical Coherence Tomography Using the RESCAN 700: Preliminary Results in Collagen Crosslinking.

Science.gov (United States)

Pahuja, Natasha; Shetty, Rohit; Jayadev, Chaitra; Nuijts, Rudy; Hedge, Bharath; Arora, Vishal

2015-01-01

To compare the penetration of riboflavin using a microscope-integrated real time spectral domain optical coherence tomography (ZEISS OPMI LUMERA 700 and ZEISS RESCAN 700) in keratoconus patients undergoing accelerated collagen crosslinking (ACXL) between epithelium on (epi-on) and epithelium off (epi-off). Intraoperative images were obtained during each of the procedures. Seven keratoconus patients underwent epi-on ACXL and four underwent epi-off ACXL. A software tool was developed using Microsoft.NET and Open Computer Vision (OpenCV) libraries for image analysis. Pre- and postprocedure images were analyzed for changes in the corneal hyperreflectance pattern as a measure of the depth of riboflavin penetration. The mean corneal hyperreflectance in the epi-on group was 12.97 ± 1.49 gray scale units (GSU) before instillation of riboflavin and 14.46 ± 2.09 GSU after AXCL (P = 0.019) while in the epi-off group it was 11.43 ± 2.68 GSU and 16.98 ± 8.49 GSU, respectively (P = 0.002). The average depth of the band of hyperreflectance in the epi-on group was 149.39 ± 15.63 microns and in the epi-off group it was 191.04 ± 32.18 microns. This novel in vivo, real time imaging study demonstrates riboflavin penetration during epi-on and epi-off ACXL.

Intraoperative Optical Coherence Tomography Using the RESCAN 700: Preliminary Results in Collagen Crosslinking

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Natasha Pahuja

2015-01-01

Full Text Available Purpose. To compare the penetration of riboflavin using a microscope-integrated real time spectral domain optical coherence tomography (ZEISS OPMI LUMERA 700 and ZEISS RESCAN 700 in keratoconus patients undergoing accelerated collagen crosslinking (ACXL between epithelium on (epi-on and epithelium off (epi-off. Methods. Intraoperative images were obtained during each of the procedures. Seven keratoconus patients underwent epi-on ACXL and four underwent epi-off ACXL. A software tool was developed using Microsoft.NET and Open Computer Vision (OpenCV libraries for image analysis. Pre- and postprocedure images were analyzed for changes in the corneal hyperreflectance pattern as a measure of the depth of riboflavin penetration. Results. The mean corneal hyperreflectance in the epi-on group was 12.97 ± 1.49 gray scale units (GSU before instillation of riboflavin and 14.46 ± 2.09 GSU after AXCL (P = 0.019 while in the epi-off group it was 11.43 ± 2.68 GSU and 16.98 ± 8.49 GSU, respectively (P = 0.002. The average depth of the band of hyperreflectance in the epi-on group was 149.39 ± 15.63 microns and in the epi-off group it was 191.04 ± 32.18 microns. Conclusion. This novel in vivo, real time imaging study demonstrates riboflavin penetration during epi-on and epi-off ACXL.

Radiation crosslinking of poly(butyl acrylate) during polymerization and grafted copolymerization with Cr(III) crosslinked collagen

International Nuclear Information System (INIS)

Pietrucha, K.; Kroh, J.

1986-01-01

Enhanced crosslinking of synthetic polymer simultaneously with grafting and homopolymerization processes has been observed in irradiated leather tanned with Cr(III) and embedded with aqueous emulsions of butyl acrylate. The extent of poly(butyl acrylate) crosslinking during copolymerization was found to be approximately one order higher than in the case of radiation polymerization of butyl acrylate in emulsion. A new method for isolation of grafted copolymer based on degradation of collagen has been developed. The extent of crosslinking was calculated from the swelling data. (author)

Collagen cross-linking in sun-exposed and unexposed sites of aged human skin

Science.gov (United States)

Yamauchi, M.; Prisayanh, P.; Haque, Z.; Woodley, D. T.

1991-01-01

A recently described nonreducible, acid-heat stable compound, histidinohydroxylysinonorleucine (HHL), is a collagen cross-link isolated from mature skin tissue. Its abundance is related to chronologic aging of skin. The present communication describes the quantity of HHL from aged human skin of the same individuals in sun-exposed (wrist) and unexposed (buttock) sites. Punch biopsies were obtained from these sites from nine people of age 60 or older. HHL contents (moles/mole of collagen) at these sites were for wrist 0.13 +/- 0.07 and for buttock 0.69 +/- 0.17 (mean +/- SD, p less than 0.001). In addition, it was found that acute irradiation of the cross-linked peptides with UVA (up to 250 J/cm2) and UVB (up to 1 J/cm2) had no effect on HHL structure. The same treatment significantly degraded another nonreducible, stable collagen cross-link, pyridinoline. The results suggest that chronic sunlight exposure may be associated with an impediment to normal maturation of human dermal collagen resulting in tenuous amount of HHL. Thus, the process of photoaging in dermal collagen is different from that of chronologic aging in human skin.

Peripheral hepatic arterial embolization with cross-linked collagen fibers

International Nuclear Information System (INIS)

Daniels, J.R.; Kerlan, R.K. Jr.; Dodds, L.; McLaughlin, P.; La Berge, J.M.; Harrington, D.; Daniels, A.M.; Ring, E.J.

1986-01-01

Hepatic artery embolization with a nonimmunogenic, cross-linked collagen preparation (Angiostat, collagen for embolization, Target Therapeutics) was studied in mongrel dogs. Flow-directed technique was used to achieve complete distal arterial occlusion. Serial liver function evaluation demonstrated marked alterations at 48 to 72 hours, partial correction at 1 week, and resolution of abnormalities by 1 month. Restoration of large-vessel blood flow was angiographically demonstrable at 1 week. Recanalization, achieved by migration of endothelial cells around the collagen, resulted in complete restoration of normal hepatic vascular and tissue anatomy at 1 month. Repeated embolization at biweekly intervals was well tolerated

Accelerated corneal collagen cross-linking in pediatric keratoconus: One year study

Directory of Open Access Journals (Sweden)

Amani E. Badawi

2017-01-01

Conclusion: Accelerated CXL shows a stabilization and beneficial clinical outcomes in pediatric keratoconus. It seems an effective and safe procedure in this age group. Effects of accelerated CXL on the posterior corneal surface will need further evaluation.

Femtosecond-assisted intrastromal corneal ring implantation for keratoconus treatment: a comparison with crosslinking combination

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Peter Alexander von Harbach Ferenczy

2015-04-01

Full Text Available Purpose: To compare visual outcomes, corneal astigmatism, and keratometric readings in patients with keratoconus who underwent intrastromal corneal ring implantation (ICRSI alone with those who underwent ICRSI combined with ultraviolet A riboflavin-mediated corneal collagen crosslinking (CXL. Methods: Pre- and post-operative best-corrected distance visual acuity (BCDVA, spherical error, cylindrical error, and mean keratometry were retrospectively compared over a period of 2 years in patients with keratoconus who underwent only ICRSI (group 1 versus those in patients who underwent combined ICRSI-CXL (group 2. Results: Thirty-two eyes of 31 patients were evaluated. CXL was performed in 10 cases (31%, and there were no complications or need for ring repositioning. BCDVA improved from 0.54 to 0.18 in the group 1 and from 0.56 to 0.17 in the group 2. Spherical and cylindrical errors and mean keratometry values significantly decreased in both groups. No patient postoperatively had visual acuity (VA of less than 20/60 on refraction, and 78% exhibited VA better than or equal to 20/40 with spectacles (72% of group 1 and 90% of group 2. Improvement in the spherical equivalent (SE value was observed in the group 1 (from -5.89 ± 3.37 preoperatively to -2.65 ± 2.65 postoperatively; p<0.05 and group 2 (from -6.91 ± 1.93 preoperatively to -2.11 ± 3.01 postoperatively; p<0.05. Conclusion: Both techniques can be considered safe and effective in improving VA and refractive SE values, in decreasing the curvature of the cone apex in the topographical analysis, and in decreasing corrected diopters postoperatively in patients with keratoconus.

Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts

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Chu, Chenyu; Deng, Jia [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Xiang, Lin; Wu, Yingying [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Wei, Xiawei [State Key Laboratory of Biotherapy and Laboratory for Aging Research, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041 (China); Qu, Yili [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Man, Yi, E-mail: manyi780203@126.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)

2016-10-01

Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

Changes in corneal sensitivity following cross-linking for progressive early-stage keratoconus

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Anelise de Medeiros Lago

2014-04-01

Full Text Available Purpose: To evaluate changes in corneal sensitivity following corneal cross-linking (CXL in patients with progressive earlier stage keratoconus. Methods: Thirty-eight eyes of 19 patients (11 women, 8 men were included in a prospective, nonrandomized clinical study. The mean patient age was 22 years (range, 18-26 years. Inclusion criteria were early stage bilateral progressive keratoconus, a transparent cornea, and a thickness of ≥440 µm in the thinnest area of the cornea. Using the Cochet-Bonnet esthesiometer, central corneal sensitivity was measured before surgery, 7 days after surgery, and once a month thereafter until recovery of baseline preoperative levels. Central corneal sensitivity >40 mm was considered normal. Results: Corneal sensitivity gradually returned to preoperative levels in all treated eyes. The mean central corneal sensitivity was 52.2, 24.0, 38.2, 42.5, 50.0, and 52.5 mm before surgery, 7 days after surgery, and at 1, 2, 3, and 4 months after surgery, respectively. Normal levels of corneal sensation, but not baseline (preoperative levels, were observed 2 months after surgery. The preoperative levels were observed 3 months after surgery. Conclusions: Our results suggest that central corneal sensitivity can be decreased for as long as 3 months after CXL for progressive earlier stage keratoconus.

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

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Lai, Jui-Yang; Ma, David Hui-Kang

2013-01-01

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

Effect of nordihydroguaiaretic acid cross-linking on fibrillar collagen: in vitro evaluation of fibroblast adhesion strength and migration

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Ana Y. Rioja

2017-04-01

Full Text Available Fixation is required to reinforce reconstituted collagen for orthopedic bioprostheses such as tendon or ligament replacements. Previous studies have demonstrated that collagen fibers cross-linked by the biocompatible dicatechol nordihydroguaiaretic acid (NDGA have mechanical strength comparable to native tendons. This work focuses on investigating fibroblast behavior on fibrillar and NDGA cross-linked type I collagen to determine if NDGA modulates cell adhesion, morphology, and migration. A spinning disk device that applies a range of hydrodynamic forces under uniform chemical conditions was employed to sensitively quantify cell adhesion strength, and a radial barrier removal assay was used to measure cell migration on films suitable for these quantitative in vitro assays. The compaction of collagen films, mediated by the drying and cross-linking fabrication process, suggests a less open organization compared to native fibrillar collagen that likely allowed the collagen to form more inter-chain bonds and chemical links with NDGA polymers. Fibroblasts strongly adhered to and migrated on native and NDGA cross-linked fibrillar collagen; however, NDGA modestly reduced cell spreading, adhesion strength and migration rate. Thus, it is hypothesized that NDGA cross-linking masked some adhesion receptor binding sites either physically, chemically, or both, thereby modulating adhesion and migration. This alteration in the cell-material interface is considered a minimal trade-off for the superior mechanical and compatibility properties of NDGA cross-linked collagen compared to other fixation approaches.

Evaluation of nanohydroxyapaptite (nano-HA) coated epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes.

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Chu, Chenyu; Deng, Jia; Man, Yi; Qu, Yili

2017-09-01

Collagen is the main component of extracellular matrix (ECM) with desirable biological activities and low antigenicity. Collagen materials have been widely utilized in guided bone regeneration (GBR) surgery due to its abilities to maintain space for hard tissue growth. However, pure collagen lacks optimal mechanical properties. In our previous study, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, with better biological activities and enhanced mechanical properties, may promote osteoblast proliferation, but their effect on osteoblast differentiation is not very significant. Nanohydroxyapatite (nano-HA) is the main component of mineral bone, which possesses exceptional bioactivity properties including good biocompatibility, high osteoconductivity and osteoinductivity, non-immunogenicity and non-inflammatory behavior. Herein, by analyzing the physical and chemical properties as well as the effects on promoting bone regeneration, we have attempted to present a novel EGCG-modified collagen membrane with nano-HA coating, and have found evidence that the novel collagen membrane may promote bone regeneration with a better surface morphology, without destroying collagen backbone. To evaluate the surface morphologies, chemical and mechanical properties of pure collagen membranes, epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes, nano-HA coated collagen membranes, nano-HA coated EGCG-collagen membranes, (ii) to evaluate the bone regeneration promoted by theses membranes. In the present study, collagen membranes were divided into 4 groups: (1) untreated collagen membranes (2) EGCG cross-linked collagen membranes (3) nano-HA modified collagen membranes (4) nano-HA modified EGCG-collagen membranes. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to evaluate surface morphologies and chemical properties, respectively. Mechanical properties were determined by differential scanning calorimeter (DSC

Effect of corneal cross-linking on contact lens tolerance in keratoconus.

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Ünlü, Metin; Yüksel, Erdem; Bilgihan, Kamil

2017-07-01

The aim was to investigate changes in corneal sensation and rigid gas-permeable (RGP) contact lens tolerance after corneal cross-linking (CXL) on patients with keratoconus. Thirty eyes of 30 patients, who were RGP lens intolerant, were treated with CXL. The main outcome measures were corneal sensation evaluation by Cochet-Bonnet esthesiometry, sub-basal nerve fibre assessment by corneal in vivo confocal microscopy and RGP contact lens tolerance evaluation with the Likert scale and wearing time. All eyes were evaluated preoperatively and post-operatively at one, three and six months after CXL procedure. The mean age was 25.3 ± 6.2 years. Preoperatively, the maximum keratometry (Kmax) in study eyes was 56.89 ± 4.60 D. Six months after CXL, it reduced to 56.03 ± 4.85 D (p = 0.01). Preoperative mean corneal sensation was 0.44 ± 0.05 g/mm 2 , (range: 0.40 to 0.55); it was significantly decreased at the first month and increased to preoperative values after six months. The sub-basal nerve plexus could not be visualised in 90 per cent of the patients by confocal microscopy at one month post-operatively. Gradual restoration of corneal innervation with almost similar preoperative levels at post-operative month six was noted. There were significant differences in Likert scores between preoperative and third and sixth months after CXL. Likert scale scores correlated significantly with corneal sensitivity. It can be concluded that increased RGP contact lens tolerance after CXL may be associated with the potential role of decreased corneal sensitivity and corneal flattening after CXL. © 2016 Optometry Australia.

Secondary cytotoxicity of (crosslinked) dermal sheep collagen during repeated exposure to human fibroblasts

NARCIS (Netherlands)

van Luyn, M.J.A.; van Wachem, P.B.; Olde damink, L.H.H.; Olde Damink, L.H.H.; Dijkstra, Pieter J.; Feijen, Jan; Nieuwenhuis, P.

1992-01-01

We investigated commercially available dermal sheep collagen either cross-linked with hexamethylenediisocyanate, or cross-linked with glutaraldehyde. In previous in vitro studies we could discriminate primary, i.e. extractable, and secondary cytotoxicity, due to cell-biomaterial interactions, i.e.

Cross-linking in collagen by nonenzymatic glycation increases the matrix stiffness in rabbit achilles tendon.

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Reddy, G Kesava

2004-01-01

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174-180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young's modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young's modulus of elasticity, and toughness (r values ranging from.61 to.94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from.22 to.84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

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Hannah J Levis

Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

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).

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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.

Multiphoton crosslinking for biocompatible 3D printing of type I collagen.

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Bell, Alex; Kofron, Matthew; Nistor, Vasile

2015-09-03

Multiphoton fabrication is a powerful technique for three-dimensional (3D) printing of structures at the microscale. Many polymers and proteins have been successfully structured and patterned using this method. Type I collagen comprises a large part of the extracellular matrix for most tissue types and is a widely used cellular scaffold material for tissue engineering. Current methods for creating collagen tissue scaffolds do not allow control of local geometry on a cellular scale. This means the environment experienced by cells may be made up of the native material but unrelated to native cellular-scale structure. In this study, we present a novel method to allow multiphoton crosslinking of type I collagen with flavin mononucleotide photosensitizer. The method detailed allows full 3D printing of crosslinked structures made from unmodified type I collagen and uses only demonstrated biocompatible materials. Resolution of 1 μm for both standing lines and high-aspect ratio gaps between structures is demonstrated and complex 3D structures are fabricated. This study demonstrates a means for 3D printing with one of the most widely used tissue scaffold materials. High-resolution, 3D control of the fabrication of collagen scaffolds will facilitate higher fidelity recreation of the native extracellular environment for engineered tissues.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

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López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Case of Medication-Resistant Acanthamoeba Keratitis Treated by Corneal Crosslinking in Turkey

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Goktug Demirci

2013-01-01

Full Text Available Purpose. To report a case of medication-resistant acanthamoeba keratitis (AK treated successfully by corneal crosslinking (CXL. Methods. A 26-year-old male with medication-resistant AK underwent a standard CXL procedure with local anesthesia, followed by central corneal epithelial debridement, application of riboflavin 0.1%, and UV-A irradiation. Results. The patient experienced a dramatic symptomatic improvement within 24 hours. At two months, keratitis was healed with a semitransparent paracentral scar that did not affect visual acuity. Conclusions. Our experience, considered in the context of recent studies, suggests that CXL may be an option for selected patients with medication-resistant AK and corneal melting. CXL allows patients to avoid emergency keratoplasty and experience rapid symptomatic relief.

Quantitative Raman characterization of cross-linked collagen thin films as a model system for diagnosing early osteoarthritis

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Wang, Chao; Durney, Krista M.; Fomovsky, Gregory; Ateshian, Gerard A.; Vukelic, Sinisa

2016-03-01

The onset of osteoarthritis (OA)in articular cartilage is characterized by degradation of extracellular matrix (ECM). Specifically, breakage of cross-links between collagen fibrils in the articular cartilage leads to loss of structural integrity of the bulk tissue. Since there are no broadly accepted, non-invasive, label-free tools for diagnosing OA at its early stage, Raman spectroscopyis therefore proposed in this work as a novel, non-destructive diagnostic tool. In this study, collagen thin films were employed to act as a simplified model system of the cartilage collagen extracellular matrix. Cross-link formation was controlled via exposure to glutaraldehyde (GA), by varying exposure time and concentration levels, and Raman spectral information was collected to quantitatively characterize the cross-link assignments imparted to the collagen thin films during treatment. A novel, quantitative method was developed to analyze the Raman signal obtained from collagen thin films. Segments of Raman signal were decomposed and modeled as the sum of individual bands, providing an optimization function for subsequent curve fitting against experimental findings. Relative changes in the concentration of the GA-induced pyridinium cross-links were extracted from the model, as a function of the exposure to GA. Spatially resolved characterization enabled construction of spectral maps of the collagen thin films, which provided detailed information about the variation of cross-link formation at various locations on the specimen. Results showed that Raman spectral data correlate with glutaraldehyde treatment and therefore may be used as a proxy by which to measure loss of collagen cross-links in vivo. This study proposes a promising system of identifying onset of OA and may enable early intervention treatments that may serve to slow or prevent osteoarthritis progression.

Biocompatibility and tissue regenerating capacity of crosslinked dermal sheep collagen

NARCIS (Netherlands)

van Wachem, P.B.; van Luyn, M.J.A.; Olde Damink, L.H.H.; Olde damink, L.H.H.; Dijkstra, Pieter J.; Feijen, Jan; Nieuwenhuis, P.

1994-01-01

The biocompatibility and tissue regenerating capacity of four crosslinked dermal sheep collagens (DSC) was studied. In vitro, the four DSC versions were found to be noncytotoxic or very low in cytoxicity. After subcutaneous implantation in rats, hexamethylenediisocyanatecrcrosslinked DSC (HDSC)

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

Science.gov (United States)

Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

2016-01-01

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

Hyperopic correction: clinical validation with epithelium-on and epithelium-off protocols, using variable fluence and topographically customized collagen corneal crosslinking

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Kanellopoulos AJ

2014-12-01

Full Text Available Anastasios John Kanellopoulos,1,2 George Asimellis1 1LaserViison.gr Clinical and Research Eye Institute, Athens, Greece; 2Department of Ophthalmology, New York University Medical School, New York, NY, USAPurpose: To report novel application of topographically-customized collagen crosslinking aiming to achieve hyperopic refractive changes. Two approaches were evaluated, one based on epithelium-off and one based on epithelium-on (transepithelial. Methods: A peripheral annular-shaped topographically customizable design was employed for high-fluence ultraviolet (UV-A irradiation aiming to achieve hyperopic refractive changes. A total of ten eyes were involved in this study. In group-A (five eyes, a customizable ring pattern was employed to debride the epithelium by excimer laser ablation, while in group-B (also five eyes, the epithelium remained intact. In both groups, specially formulated riboflavin solutions were applied. Visual acuity, cornea clarity, keratometry, topography, and pachymetry with a multitude of modalities, as well as endothelial cell counts were evaluated. Results: One year postoperatively, the following changes have been noted: in group-A, average uncorrected distance visual acuity changed from 20/63 to 20/40. A mean hyperopic refractive increase of +0.75 D was achieved. There was some mild reduction in the epithelial thickness. In group-B, average uncorrected distance visual acuity changed from 20/70 to 20/50. A mean hyperopic refractive increase of +0.85 D was achieved. Epithelial thickness returned to slightly reduced levels (compared to baseline in group-A, whereas to slightly increased levels in group-B. Conclusion: We introduce herein the novel application of a topographically-customizable collagen crosslinking to achieve a hyperopic refractive effect. This novel technique may be applied either with epithelial removal, offering a more stable result or with a non-ablative and non-incisional approach, offering a minimally

Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.

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Chen, Vicki M; Shelke, Rajani; Nyström, Alexander; Laver, Nora; Sampson, James F; Zhiyi, Cao; Bhat, Najma; Panjwani, Noorjahan

2018-06-16

Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The immune-histologic and morphologic changes in the corneas of the mouse model for this disease have not been described in the literature. Our purpose is to characterize the eyes of these mice to determine if this is an appropriate model for study of human therapeutics. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess the relative collagen VII protein levels and its location within the cornea. Additional IHC for inflammatory and fibrotic biomarkers alpha-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting opacification of the corneas of animals of differing ages were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. IHC and WB confirmed that these mice are deficient in collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-β showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-β appear to be

SECONDARY CYTOTOXICITY OF CROSS-LINKED DERMAL SHEEP COLLAGENS DURING REPEATED EXPOSURE TO HUMAN FIBROBLASTS

NARCIS (Netherlands)

VANLUYN, MJA; VANWACHEM, PB; DAMINK, LHHO; DIJKSTRA, PJ; FEIJEN, J; NIEUWENHUIS, P

1992-01-01

We investigated commercially available dermal sheep collagen either cross-linked with hexamethylenedlisocyanate, or cross-linked with glutaraldehyde. In previous in vitro studies we could discriminate primary, i.e. extractable, and secondary cytotoxicity, due to cell-biomaterial interactions, i.e.

Changes in Cytokines and Aggrecan ARGS Neoepitope in Synovial Fluid and Serum and in C-Terminal Crosslinking Telopeptide of Type II Collagen and N-Terminal Crosslinking Telopeptide of Type I Collagen in Urine Over Five Years After Anterior Cruciate Ligament Rupture

DEFF Research Database (Denmark)

Struglics, André; Larsson, Staffan; Kumahashi, Nobuyuki

2015-01-01

OBJECTIVE: To prospectively monitor levels of proinflammatory cytokines and aggrecan ARGS neoepitope in synovial fluid and serum as well as levels of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and N-terminal crosslinking telopeptide of type I collagen (NTX-I) in urine after ...

[The status quo and expectation of corneal research in China].

Science.gov (United States)

Shi, Weiyun; Xie, Lixin

2014-09-01

In China, corneal disease is currently the second leading cause of blindness. Severe donor shortage, insufficient technique supports and promotion, and the lack of corneal disease specialists due to poor systematic training are all urgent problems to be resolved. The last 5 years have witnessed a considerable progress in basic and clinical researches of corneal disease. Investigations on the pathogenesis and treatment of fungal keratitis have won an international reputation. Results from the study of corneal reconstruction with tissue-engineered and acellular matrix corneas have been tested in clinical trials with good preliminary performance. Moreover, the clinical researches of corneal refractive surgery have kept pace with the latest international progresses. However, Descemet's membrane endothelial keratoplasty needs further promotion, and the development and application of keratoprosthesis remains a blank. Although keratoprosthesis and corneal collagen cross-linking have been widely applied in Europe with satisfactory clinical efficacy, they are still under assessment by China Food and Drug Administration for approval of use.

Quantification of collagen ultrastructure after penetrating keratoplasty - implications for corneal biomechanics.

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Craig Boote

Full Text Available To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK, and evaluate their possible implications for corneal biomechanics.A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left/28 (right years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal×0.25 mm (vertical intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas.Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident.Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

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Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

2014-06-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

Corneal Crosslinking With Rose Bengal and Green Light: Efficacy and Safety Evaluation.

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Zhu, Hong; Alt, Clemens; Webb, Robert H; Melki, Samir; Kochevar, Irene E

2016-09-01

To evaluate crosslinking of cornea in vivo using green light activation of Rose Bengal (RGX) and assess potential damaging effects of the green light on retina and iris. Corneas of Dutch belted rabbits were de-epithelialized, then stained with Rose Bengal and exposed to green light, or not further treated. Corneal stiffness was measured by uniaxial tensiometry. Re-epithelialization was assessed by fluorescein fluorescence. Keratocytes were counted on hematoxylin and eosin (H&E)-stained sections, and iris cell damage was assessed by lactate dehydrogenase staining. Thermal effects on the blood-retinal barrier (BRB) were assessed by fluorescein angiography and those on photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris by light microscopy and transmission electron microscopy. RGX (10-min irradiation; 150 J/cm) increased corneal stiffness 1.9-fold on day 1 (1.25 ± 0.21 vs. 2.38 ± 0.59 N/mm; P = 0.036) and 2.8-fold compared with controls on day 28 (1.70 ± 0.74 vs. 4.95 ± 1.86 N/mm; P = 0.003). Keratocytes decreased only in the anterior stroma on day 1 (24.0 ± 3.0 vs. 3.67 ± 4.73, P = 0.003) and recovered by day 28 (37.7 ± 8.9 vs. 34.5 ± 2.4, P = 0.51). Iris cells were not thermally damaged. No evidence of BRB breakdown was detected on days 1 or 28. Retina from RGX-treated eyes seemed normal with RPE cells showing intact nuclei shielded apically by melanosomes, morphologically intact photoreceptor outer segments, normal outer nuclear layer thickness, and choriocapillaris containing intact erythrocytes. The substantial corneal stiffening produced by RGX together with the lack of significant effects on keratocytes and no evidence for retina or iris damage suggest that RGX-initiated corneal crosslinking may be a safe, rapid, and effective treatment.

Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy (Conference Presentation)

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Kwok, Sheldon J. J.; Kuznetsov, Ivan A.; Kim, Moonseok; Choi, Myunghwan; Scarcelli, Giuliano; Yun, Seok-Hyun

2017-02-01

Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively. Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.

Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

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Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

2016-02-01

Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

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Wayne A Cabral

2014-06-01

Full Text Available Cyclophilin B (CyPB, encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO mice are small, with reduced femoral areal bone mineral density (aBMD, bone volume per total volume (BV/TV and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11% collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1 activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

Science.gov (United States)

Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

2014-01-01

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

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Lai JY

2013-10-01

Full Text Available Jui-Yang Lai,1–3 David Hui-Kang Ma4,5 1Institute of Biochemical and Biomedical Engineering, 2Biomedical Engineering Research Center, 3Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; 4Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 5Department of Chinese Medicine, Chang Gung University, Taoyuan, Taiwan Abstract: Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA cross-linked amniotic membrane (AM on limbal epithelial cell (LEC cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous

Characterization of elasticity and hydration of composite hydrogel based on collagen-iota carrageenan as a corneal tissue engineering

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Rinawati, M.; Triastuti, J.; Pursetyo, K. T.

2018-04-01

The cornea is a refractive element of the eye that serves to continue the stimulation of light into the eye it has a clear, transparent, elastic and relatively thick tissue. Factors caused corneal blindness, are dystrophy, keratoconus, corneal scaring. Hydrogels can be made from polysaccharide derivatives that have gelation properties such as iota carrageenan. Therefore, it is a need to develop composite hydrogel based collagen-iota carragenan as an engineeried corneal tissue with high elasticity and hydration properties. Collagen hydrogel has a maximum water content an has equlibrium up to 40 %, less than the human cornea, 81 % and under normal hydration conditions, the human cornea can transmit 87 % of visible light. In addition, the refractive index on the surface of the cornea with air is 1.375-1.380. Based on this study, it is necessary to conduct research on the development and composition of hydrogel composite collagen-iota carrageen hydrogen based on. The best result was K5 (5:5) treatment, which has the equilibrium water content of 87.07 % and viscosity of 10.7346 Pa.s.

Quantification of Collagen Ultrastructure after Penetrating Keratoplasty – Implications for Corneal Biomechanics

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Gardner, Steven J.; Kamma-Lorger, Christina S.; Hayes, Sally; Nielsen, Kim; Hjortdal, Jesper; Sorensen, Thomas; Terrill, Nicholas J.; Meek, Keith M.

2013-01-01

Purpose To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics. Methods A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas. Results Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident. Conclusions Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the

Effect of the Regenerative Agent Poly(Carboxymethylglucose Sulfate) on Corneal Wound Healing After Corneal Cross-Linking for Keratoconus.

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Kymionis, George D; Liakopoulos, Dimitrios A; Grentzelos, Michael A; Tsoulnaras, Konstantinos I; Detorakis, Efstathios T; Cochener, Béatrice; Tsilimbaris, Miltiadis K

2015-08-01

To evaluate the effect of a regenerative agent (RGTA) [Cacicol20-poly(carboxymethyl glucose sulfate); OTR3, Paris, France] on corneal reepithelialization and pain after corneal cross-linking (CXL) for keratoconus. In this prospective comparative (contralateral) clinical study, patients with bilateral progressive keratoconus underwent CXL treatment. The corneal epithelium during CXL was removed using transepithelial phototherapeutic keratectomy (Cretan protocol). One eye of each patient was randomly instilled with an RGTA (Cacicol20) once a day (study group), whereas the fellow eye was instilled with artificial tears (control group). Patients were examined daily until complete reepithelialization. Postoperative examinations included slit-lamp biomicroscopy to assess the epithelial defect size and subjective evaluation of pain. The study enrolled 18 patients (36 eyes). The mean epithelial defect size for study and control groups was 19.6 ± 4.2 mm versus 21.5 ± 2.8 mm, respectively, at day 1 (P = 0.019) and 6.4 ± 3.4 mm versus 7.9 ± 4.3 mm, respectively, at day 2 (P = 0.014). At day 3 postoperatively, 61.1% of study eyes were fully reepithelialized, compared with 11.1% of control eyes (P = 0.002). RGTA (Cacicol20) instillation seems to result in faster corneal reepithelialization after CXL in this study. However, there was no significant effect in subjective pain/discomfort.

Impact of corneal cross-linking combined with photorefractive keratectomy on blurring strength

OpenAIRE

Labiris, Georgios; Sideroudi, Haris; Angelonias, Dimitris; Georgantzoglou, Kimonas; Kozobolis, Vassilios P

2016-01-01

Georgios Labiris,1,2 Haris Sideroudi,2 Dimitris Angelonias,2 Kimonas Georgantzoglou,2 Vassilios P Kozobolis1,21Department of Ophthalmology, University Hospital of Alexandroupolis, 2Eye Institute of Thrace, Alexandroupolis, GreecePurpose: The aim of this study was to evaluate the impact of corneal cross-linking combined with photorefractive keratectomy (PRK) on blurring strength.Methods: A total of 63 patients with keratoconus were recruited for this study, and two study groups were formed acc...

Mechanical properties and collagen cross-linking of the patellar tendon in old and young men

DEFF Research Database (Denmark)

Couppé, C; Hansen, P; Kongsgaard, M

2009-01-01

were higher in OM than in YM (73 +/- 13 vs. 11 +/- 2 mmol/mol; P appreciably influence the dimensions or mechanical properties of the human patellar tendon in vivo. Collagen concentration was reduced, whereas both enzymatic......Age-related loss in muscle mass and strength impairs daily life function in the elderly. However, it remains unknown whether tendon properties also deteriorate with age. Cross-linking of collagen molecules provides structural integrity to the tendon fibrils and has been shown to change with age...... in animals but has never been examined in humans in vivo. In this study, we examined the mechanical properties and pyridinoline and pentosidine cross-link and collagen concentrations of the patellar tendon in vivo in old (OM) and young men (YM). Seven OM (67 +/- 3 years, 86 +/- 10 kg) and 10 YM (27 +/- 2...

Automated Detection and Classification of Corneal Haze Using Optical Coherence Tomography in Patients With Keratoconus After Cross-Linking.

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Dhaini, Ahmad R; Abdul Fattah, Maamoun; El-Oud, Sara Maria; Awwad, Shady T

2018-03-13

To evaluate a proposed technology for offering objective grading and mapping of corneal haze as detected by corneal spectral domain optical coherence tomography after corneal cross-linking. This was a retrospective study to evaluate corneal optical coherence tomography images performed on 44 eyes of 44 patients who underwent corneal cross-linking between January 2014 and May 2015, at the American University of Beirut Medical Center. Overall average brightness of the cornea was markedly increased from 43.4% (±6.0) at baseline to 50.2% (±4.4) at 1 month, 47.9% (±4.4) at 3 months, and 46.4% (±5.7) at 6 months with P <0.001, <0.001, and 0.005, respectively. In the anterior stroma, the average brightness significantly increased at 1, 3, and 6 months with values of 54.8% (±3.9), 52.5% (±5.2), and 49.7% (±6.9) with P <0.001, <0.001, and 0.003, respectively. In the mid stroma, the change was clinically significant at 1 and 3 months, whereas in the posterior stroma, it was only significant at 1 month compared with baseline (P = 0.003). Overall, haze was mostly present at 1 month after surgery in all regions, especially in the anterior (32.1%; ±19.2) and mid stromal regions (9.1%; ±18.8), P <0.001 and 0.001, respectively. In contrast, haze in the posterior stromal region peaks at 3 and 6 months after surgery. Anterior stromal haze was the greatest in intensity and area and it was present for a longer time span than mid and posterior stromal haze. At 12 months, the anterior stroma had still more haze intensity than preoperatively. This image-based software can provide objective and valuable quantitative measurements of corneal haze, which may impact clinical decision-making after different corneal surgeries.

Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.

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Takezawa, Toshiaki; Nishikawa, Kazunori; Wang, Pi-Chao

2011-09-01

The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test. Copyright © 2011 Elsevier Ltd. All rights reserved.

Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization.

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Tidu, Aurélien; Ghoubay-Benallaoua, Djida; Teulon, Claire; Asnacios, Sophie; Grieve, Kate; Portier, François; Schanne-Klein, Marie-Claire; Borderie, Vincent; Mosser, Gervaise

2018-05-29

This study aimed at controlling both the organization and the transparency of dense collagen scaffolds making use of the lyotropic mesogen properties of collagen. Cholesteric or plywood-like liquid crystal phases were achieved using mixtures of acetic and hydrochloric acids as solvents. The critical pH at which the switch between the two phases occurred was around pH = 3. The use of the two acids led to fibrillated collagen I scaffolds, whose visual aspect ranged from opaque to transparent. Rheological investigations showed that viscoelastic properties of the plywood-like solutions were optimized for molding due to faster recovery. They also confirmed the correlation between the elastic modulus and the diameter of collagen fibrils obtained after fibrillogenesis under ammonia vapor. Human corneal epithelial cells, grown from donor limbal explants, were cultured both on transparent plywood-like matrices and on human amniotic membranes for 14 days. The development of corneal epithelium and the preservation of epithelial stem cells were checked by optical microscopy, colony formation assay, immuno-fluorescence and quantitative polymerase chain reaction. A higher level of amplification of limbal stem cells was obtained with collagen matrices compared with amniotic membranes, showing the high biocompatibility of our scaffolds. We therefore suggest that collagen solutions presenting both plywood-like organization and transparency might be of interest for biomedical applications in ophthalmology.

Corneal changes following collagen cross linking and simultaneous topography guided photoablation with collagen cross linking for keratoconus

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Prema Padmanabhan

2014-01-01

Full Text Available Purpose: To compare the outcome of Collagen cross-linking (CXL with that following topography-guided customized ablation treatment (T-CAT with simultaneous CXL in eyes with progressive keratoconus. Materials and Methods: This was a prospective, non-randomized single centre study of 66 eyes with progressive keratoconus. Of these, 40 eyes underwent CXL and 26 eyes underwent T-CAT + CXL. The refractive, topographic, tomographic and aberrometric changes measured at baseline, 1, 3 and 6 months post-operatively were compared between both groups. Results: After a mean follow-up of 7.7 ± 1.3 months, the mean retinoscopic cylinder decreased by 1.02 ± 3.16 D in the CXL group ( P = 0.1 and 2.87 ± 3.22 D in the T-CAT + CXL group ( P = 0.04. The Best corrected visual acuity increased by 2 lines or more in 10% of eyes in the CXL group and in 23.3% of eyes in the T-CAT + CXL group. The mean steepest-K reduced by 0.40 ± 3.71 D ( P = 0.77 in the CXL group and by 2.91 ± 2.01D ( P = 0.03 in the T-CAT + CXL group. The sag factor and surface asymmetry index showed no significant change in the CXL group but reduced by 3.59 ± 5.94 D ( P = 0.01 and 0.72 ± 1.18 ( P = 0.02 respectively in the T-CAT + CXL group. There was a significant increase in the highest posterior corneal elevation in both groups (9.57 ± 14.93 μ in the CXL group and 7.85 ± 9.25 μ in the T-CAT + CXL group, P ≤ 0.001 for both. There was significantly greater reduction of mean coma ( P < 0.001 and mean higher-order aberrations ( P = 0.01 following T-CAT + CXL compared to CXL. Conclusions: CAT + CXL is an effective approach to confer biomechanical stability and to improve the corneal contour in eyes with keratoconus and results in better refractive, topographic and aberrometric outcomes than CXL alone.

Comparative study of long-term outcomes of accelerated and conventional collagen crosslinking for progressive keratoconus.

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Males, J J; Viswanathan, D

2018-01-01

PurposeTo compare the long-term outcomes of accelerated corneal collagen crosslinking (CXL) to conventional CXL for progressive keratoconus.Patients and methodsComparative clinical study of consecutive progressive keratoconic eyes that underwent either accelerated CXL (9 mW/cm 2 ultraviolet A (UVA) light irradiance for 10 min) or conventional CXL (3 mW/cm 2 UVA light irradiance for 30 min). Eyes with minimum 12 months' follow-up were included. Post-procedure changes in keratometry readings (Flat meridian: K1; steep meridian: K2), central corneal thickness (CCT), best spectacle-corrected visual acuity (BSCVA), and manifest refraction spherical equivalent (MRSE) were analysed.ResultsA total of 42 eyes were included. In all, 21 eyes had accelerated CXL (20.5±5.5 months' follow-up) and 21 eyes had conventional CXL group (20.2±5.6 months' follow-up). In the accelerated CXL group, a significant reduction in K2 (P=0.02), however no significant change in K1 (P=0.35) and CCT (P=0.62) was noted. In the conventional CXL group, a significant reduction was seen in K1 (P=0.01) and K2 (P=0.04), but not in CCT (P=0.95). Although both groups exhibited significant reductions in K2 readings, no noteworthy differences were noted between them (P=0.36). Improvements in BSCVA (accelerated CXL; P=0.22 and conventional CXL; P=0.20) and MRSE (accelerated CXL; P=0.97 and conventional CXL; P=0.54) were noted, however were not significant in either group.ConclusionAccelerated and conventional CXL appear to be effective procedures for stabilising progressive keratoconus in the long-term.

Simultaneous versus Sequential Accelerated Corneal Collagen Cross-Linking and Wave Front Guided PRK for Treatment of Keratoconus: Objective and Subjective Evaluation.

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Abou Samra, Waleed Ali; El Emam, Dalia Sabry; Farag, Rania Kamel; Abouelkheir, Hossam Youssef

2016-01-01

Aim . To compare objective and subjective outcome after simultaneous wave front guided (WFG) PRK and accelerated corneal cross-linking (CXL) in patients with progressive keratoconus versus sequential WFG PRK 6 months after CXL. Methods . 62 eyes with progressive keratoconus were divided into two groups; the first including 30 eyes underwent simultaneous WFG PRK with accelerated CXL. The second including 32 eyes underwent subsequent WFG PRK performed 6 months later after accelerated CXL. Visual, refractive, topographic, and aberrometric data were determined preoperatively and during 1-year follow-up period and the results compared in between the 2 studied groups. Results . All evaluated visual, refractive, and aberrometric parameters demonstrated highly significant improvement in both studied groups (all P PRK and accelerated CXL is an effective and safe option to improve the vision in mild to moderate keratoconus. In one-year follow-up, there is no statistically significant difference between the simultaneous and sequential procedure.

Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

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Janina Jamasbi, RPh

2016-04-01

Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model.

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Gore, Daniel M; O'Brart, David; French, Paul; Dunsby, Chris; Allan, Bruce D

2015-07-01

To measure depth-specific riboflavin concentrations in corneal stroma using two-photon fluorescence microscopy and compare commercially available transepithelial corneal collagen cross-linking (CXL) protocols. Transepithelial CXL riboflavin preparations--MedioCross TE, Ribocross TE, Paracel plus VibeX Xtra, and iontophoresis with Ricrolin+--were applied to the corneal surface of fresh postmortem rabbit eyes in accordance with manufacturers' recommendations for clinical use. Riboflavin 0.1% (VibeX Rapid) was applied after corneal epithelial debridement as a positive control. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35-μm thick were cut on a cryostat, mounted on a slide, and imaged by two-photon fluorescence microscopy. Mean (SD) concentrations were calculated from five globes tested for each protocol. Peak riboflavin concentration of 0.09% (± 0.01) was observed within the most superficial stroma (stromal depth 0-10 μm) in positive controls (epithelium-off). At the same depth, peak stromal riboflavin concentrations for MedioCross TE, Ricrolin+, Paracel/Xtra, and Ribocross TE were 0.054% (± 0.01), 0.031% (0.003), 0.021% (± 0.001), and 0.015% (± 0.004), respectively. At a depth of 300 μm (within the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (± 0.006), while at the same depth MedioCross TE and Ricrolin+ achieved 0.018% (± 0.006) and 0.016% (0.002), respectively. None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300-μm depth. Overall, MedioCross TE was the best-performing transepithelial formulation. Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. Existing commercial transepithelial CXL protocols achieve relatively low riboflavin concentrations in the anterior corneal stroma when compared to gold standard epithelium-off absorption

Age-related effect on the concentration of collagen crosslinks in human osteonal and interstitial bone tissue.

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Nyman, Jeffry S; Roy, Anuradha; Acuna, Rae L; Gayle, Heather J; Reyes, Michael J; Tyler, Jerrod H; Dean, David D; Wang, Xiaodu

2006-12-01

Collagen crosslinks are important to the quality of bone and may be contributors to the age-related increase in bone fracture. This study was performed to investigate whether age and gender effects on collagen crosslinks are similar in osteonal and interstitial bone tissues. Forty human cadaveric femurs were collected and divided into two age groups: middle-aged (42-63 years of age) and elderly (69-90 years of age) with ten males and ten females in each group (n = 10). Micro-cores of bone tissue from both secondary osteons and interstitial regions in the medial quadrant of the diaphysis were extracted using a custom-modified, computer-controlled milling machine. The bone specimens were then analyzed using high performance liquid chromatography to determine the effects of age and gender on the concentration of mature, enzymatic crosslinks (hydroxylysyl-pyridinoline-HP and lysyl-pyridinoline-LP) and a non-enzymatic crosslink (pentosidine-PE) at these two microstructural sites. The results indicate that age has a significant effect on the concentration of LP and PE, while gender has a significant effect on HP and LP. In addition, the concentration of the crosslinks in the secondary osteons is significantly different from that in the interstitial bone regions. These results suggest that the amount of non-enzymatic crosslinking may increase while that of mature enzymatic crosslinking may decrease with age. Such changes could potentially reduce the inherent quality of the bone tissue in the elderly skeleton.

Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications.

Science.gov (United States)

Rizwan, Muhammad; Peh, Gary S L; Ang, Heng-Pei; Lwin, Nyein Chan; Adnan, Khadijah; Mehta, Jodhbir S; Tan, Wui Siew; Yim, Evelyn K F

2017-03-01

Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with diverse tissue growth applications. We hypothesize that, with suitable material modifications, GelMA could be employed for growth and implantation of tissue-engineered human corneal endothelial cell (HCEC) monolayer. Tissue-engineered HCEC monolayer could potentially be used to treat corneal blindness due to corneal endothelium dysfunction. Here, we exploited a sequential hybrid (physical followed by UV) crosslinking to create an improved material, named as GelMA+, with over 8-fold increase in mechanical strength as compared to regular GelMA. The presence of physical associations increased the subsequent UV-crosslinking efficiency resulting in robust materials able to withstand standard endothelium insertion surgical device loading. Favorable biodegradation kinetics were also measured in vitro and in vivo. We achieved hydrogels patterning with nano-scale resolution by use of oxygen impermeable stamps that overcome the limitations of PDMS based molding processes. Primary HCEC monolayers grown on GelMA+ carrier patterned with pillars of optimal dimension demonstrated improved zona-occludin-1 expression, higher cell density and cell size homogeneity, which are indications of functionally-superior transplantable monolayers. The hybrid crosslinking and fabrication approach offers potential utility for development of implantable tissue-engineered cell-carrier constructs with enhanced bio-functional properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma

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Saud A. Alanazi

2015-01-01

Full Text Available We report here the ultrastructural organization of collagen fibrils (CF and proteoglycans (PGs of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS/dermatan sulfate (DS PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark.

Regulation of corneal stroma extracellular matrix assembly.

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Chen, Shoujun; Mienaltowski, Michael J; Birk, David E

2015-04-01

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Influence of oxazolidines and zirconium oxalate crosslinkers on the hydrothermal, enzymatic, and thermo mechanical stability of type 1 collagen fiber

International Nuclear Information System (INIS)

Haroun, Mahdi A.; Khirstova, Palmina K.; Gasmelseed, Gurashi A.; Covington, Antony D.

2009-01-01

Stabilization of type I rat tail tendon (RTT) collagen by crosslink agent oxazolidine and zirconium oxalate was studied to understand the effect on the thermal, enzymatic and mechanical stability of collagen. The results show that both oxazolidine and zirconium oxalate imparts thermal stability to collagen, and oxazolidine exhibits a marked increase in the peak temperature and enthalpy changes when compared to both native and zirconium oxalate tanned RTT. There is a decrease in the peak temperature and the enthalpy changes of oxazolidine tanned RTT fibers after treatment with urea, suggesting the possibility of alterations in the secondary structure of collagen after tanning. Oxazolidine, which forms carbocationic intermediates species in solution, have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium, which largely forms monomeric species in solution. Zirconium does not seem to change the tensile strength of RTT fibers significantly in wet condition as well as oxazolidine

Influence of oxazolidines and zirconium oxalate crosslinkers on the hydrothermal, enzymatic, and thermo mechanical stability of type 1 collagen fiber

Energy Technology Data Exchange (ETDEWEB)

Haroun, Mahdi A. [Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM, Serdang (Malaysia)], E-mail: Mahdiupm@hotmail.com; Khirstova, Palmina K. [People' s Hall 11113, P.O. Box 6272, Khartoum (Sudan); Gasmelseed, Gurashi A. [Juba University, Leather Dept. P.O. Box 12327 Khartoum (Sudan); Covington, Antony D. [Leather Centre, University College Northampton, Northampton (United Kingdom)

2009-02-20

Stabilization of type I rat tail tendon (RTT) collagen by crosslink agent oxazolidine and zirconium oxalate was studied to understand the effect on the thermal, enzymatic and mechanical stability of collagen. The results show that both oxazolidine and zirconium oxalate imparts thermal stability to collagen, and oxazolidine exhibits a marked increase in the peak temperature and enthalpy changes when compared to both native and zirconium oxalate tanned RTT. There is a decrease in the peak temperature and the enthalpy changes of oxazolidine tanned RTT fibers after treatment with urea, suggesting the possibility of alterations in the secondary structure of collagen after tanning. Oxazolidine, which forms carbocationic intermediates species in solution, have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium, which largely forms monomeric species in solution. Zirconium does not seem to change the tensile strength of RTT fibers significantly in wet condition as well as oxazolidine.

A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells.

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Dimitrios Karamichos

Full Text Available Human corneal fibroblasts (HCF and corneal stromal stem cells (CSSC each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7. Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Stromal demarcation line induced by corneal cross-linking in eyes with keratoconus and nonkeratoconic asymmetric topography.

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Malta, João B N; Renesto, Adimara C; Moscovici, Bernardo K; Soong, H K; Campos, Mauro

2015-02-01

To evaluate stromal demarcation lines following corneal cross-linking (CXL) using anterior segment optical coherence tomography in patients with keratoconus and nonkeratoconic asymmetric topography. Fifth-nine eyes of 59 patients were enrolled in a retrospective comparative case series, of which 19 eyes had keratoconus and 40 eyes had asymmetric topography. Eyes with asymmetric topography were treated in preparation for photorefractive keratectomy. One month after CXL, a stromal demarcation line was evaluated at 5 standardized corneal points using anterior segment optical coherence tomography. Mean stromal demarcation line depths were measured at 5 points on the cornea, namely, centrally, 3.0 mm temporally, 1.5 mm temporally, 3.0 mm nasally, and 1.5 mm nasally. For the keratoconus group, the values were 178 ± 47, 123 ± 15, 152 ± 47, 125 ± 23, and 160 ± 43 μm, respectively. For the asymmetric corneal topography group (without keratoconus), they were 305 ± 64, 235 ± 57, 294 ± 50, 214 ± 54, and 285 ± 58 μm, respectively. There was no correlation between central corneal pachymetry and stromal demarcation line depth in all 5 measured corneal points in both groups. CXL treatment profiles are similar in keratoconic and nonkeratoconic eyes with asymmetric topography.

An age-related study of morphology and cross-link composition of collagen fibrils in the digital flexor tendons of young thoroughbred horses.

Science.gov (United States)

Patterson-Kane, J C; Parry, D A; Birch, H L; Goodship, A E; Firth, E C

1997-01-01

The superficial digital flexor tendon is the most commonly injured tendon in the racing Thoroughbred. Despite the clinical significance of this structure, only limited data exist regarding normal age-related morphology of the tensile units, the collagen fibrils. The age at which these collagen fibrils become mature in composition and structure may be of importance. Consequently, the association of age and collagen fibril crosslink composition, diameter distribution and crimp morphology in the superficial and deep digital flexor tendons of Thoroughbreds up to and including three years of age has been studied. Replacement of immature crosslinks, peaking of the collagen fibril mass-average diameter and collagen fibril index, and stabilization of collagen crimp morphology changes supported the hypothesis that both digital flexor tendons become mature in structure by two years of age.

Evaluation of the shifting of the line of sight and higher order aberrations of eyes with keratoconus after corneal cross-linking.

Science.gov (United States)

Kosekahya, Pinar; Koc, Mustafa; Tekin, Kemal; Uzel, Murat; Atilgan, Cemile Ucgul; Caglayan, Mehtap; Yilmazbas, Pelin

2017-10-01

To evaluate changes in the coordinates of the line of sight (LoS) and higher order aberrations (HOAs) of eyes with keratoconus, following corneal cross-linking (CXL). All patients (93 eyes) underwent detailed ophthalmologic examination and Pentacam HR measurements at baseline and at 3, 6, and 12 months after corneal CXL. LoS coordinates on the horizontal (x) and vertical (y) axes, vertical coma, vertical trefoil, spherical aberration, total root-mean square (RMS), and HOA-RMS values were recorded along with visual acuity and topographical parameters. LoS significantly shifted to the nasal region after corneal CXL in both right and left eyes (p=0.003 and p=0.01, respectively). Horizontal axis values of both eyes significantly shifted to the temporal region at postoperative 6th months compared to the baseline measurements (p=0.02 and p=0.02, respectively) and remained the same between postoperative 6th months and 12th months (p=1.00 and p=0.97, respectively). Total-RMS, HOA-RMS, vertical coma, and spherical aberration values significantly improved after corneal CXL (pbaseline measurements (p=0.003, p=0.02, p0.05 for all values). The changes in horizontal-axis coordinates in left eyes were significantly correlated with anterior elevation, anterior astigmatism, total-RMS and HOA-RMS changes (r 2 =0.20, p=0.03; r 2 =0.35, pshifted to the nasal region and HOAs improved after corneal CXL and these changes stabilized 6th months after corneal CXL. It would be more better to perform refractive surgery in crosslinked corneas at least 6th months after corneal CXL. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Topography-guided transepithelial PRK after intracorneal ring segments implantation and corneal collagen CXL in a three-step procedure for keratoconus.

Science.gov (United States)

Coskunseven, Efekan; Jankov, Mirko R; Grentzelos, Michael A; Plaka, Argyro D; Limnopoulou, Aliki N; Kymionis, George D

2013-01-01

To present the results of topography-guided transepithelial photorefractive keratectomy (PRK) after intracorneal ring segments implantation followed by corneal collagen cross-linking (CXL) for keratoconus. In this prospective case series, 10 patients (16 eyes) with progressive keratoconus were included. All patients underwent topography-guided transepithelial PRK after Keraring intracorneal ring segments (Mediphacos Ltda) implantation, followed by CXL treatment. The follow-up period was 6 months after the last procedure for all patients. Time interval between both intracorneal ring segments implantation and CXL and between CXL and topography-guided transepithelial PRK was 6 months. LogMAR mean uncorrected distance visual acuity and mean corrected distance visual acuity were significantly improved (PPRK with intracorneal ring segments implantation and CXL in a three-step procedure seems to be an effective, promising treatment sequence offering patients a functional visual acuity and ceasing progression of the ectatic disorder. A longer follow-up and larger case series are necessary to thoroughly evaluate safety, stability, and efficacy of this innovative procedure. Copyright 2013, SLACK Incorporated.

Technical advances in the sectioning of dental tissue and of on-section cross-linked collagen detection in mineralized teeth.

Science.gov (United States)

Singhrao, Sim K; Sloan, Alastair J; Smith, Emma L; Archer, Charles W

2010-08-01

Immunohistochemical detection of cross-linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on-section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New. It was possible to cut thin (1 mum) sections of mineralized teeth, and immunofluorescence characterization of cross-linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was "patchy" and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on-section protein detection but importantly for detecting cross-linked fibrous collagens in both soft and mineralized tissue sections.

Disentangling mechanisms involved in collagen pyridinoline cross-linking : The immunophilin FKBP65 is critical for dimerization of lysyl hydroxylase 2

NARCIS (Netherlands)

Gjaltema, Rutger A. F.; van der Stoel, Miesje M.; Boersema, Miriam; Bank, Ruud A.

2016-01-01

Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS

Comparison of the Early Clinical Outcomes between Combined Small-Incision Lenticule Extraction and Collagen Cross-Linking versus SMILE for Myopia.

Science.gov (United States)

Ng, Alex L K; Chan, Tommy C Y; Cheng, George P M; Jhanji, Vishal; Ye, Cong; Woo, Victor C P; Lai, Jimmy S M

2016-01-01

Background. To compare the early outcome of combined SMILE and collagen crosslinking (SMILE Xtra) with SMILE. Method. Prospective, comparative interventional study of 21 eyes receiving SMILE Xtra using a low energy protocol and 32 control eyes receiving SMILE only. The outcomes were compared at 1, 3, and 6 months postoperatively. Results. Both groups had myopia with spherical equivalent refraction (SEQ) > 4.00 D. The SMILE Xtra group had thinner preoperative central corneal thickness and residual stromal bed thickness (p line in corrected distance visual acuity. The safety index was 0.96 ± 0.06 and 1.00 ± 0.00 in SMILE Xtra and control, respectively (p SMILE Xtra and +0.03 ± 0.25 D for control (p = 0.021). The efficacy index was 0.88 ± 0.13 and 0.97 ± 0.06, respectively (p = 0.005). Conclusion. SMILE Xtra had good overall safety profile and predictability at 6 months. However, when compared with control, the safety index and efficacy index were statistically significantly lower in the early postoperative period.

[Dynamics of riboflavin level in aqueous humour of anterior chamber of experimental animals under standard stroma saturation by ultraviolet corneal cross-linking solutions].

Science.gov (United States)

Bikbov, M M; Shevchuk, N E; Khalimov, A R; Bikbova, G M

To evaluate the dynamics of riboflavin changes in the aqueous humour of the anterior chamber (AHAC) of rabbits' eyes during standard ultraviolet (UV) cross-linking with account to the area of corneal debridement. Forty two rabbits were studied sequentially. The following solutions of riboflavin were used for cornea saturation: IR - 0.1% isosmotic riboflavin, D - Dextralink (0.1% riboflavin with 20% dextran), R - 0.1% riboflavin with 1.0% hydroxypropylmethylcellulose (HPMC). Each solution was evaluated in 3 groups that differed in the diameter of corneal debridement: group 1 - Epi-Off 3 mm (IR-3, D-3, P-3), group 2 - Epi-Off 6 mm (IR-6, D-6, R-6), and group 3 - Epi-Off 9 mm (IR-9, D-9, R-9). Aqueous humour sampling (252 samples in total) was performed in 10-minute intervals within a 60 minute period. Riboflavin levels were measured by enzyme-linked immunoassay (ID-Vit microbiological test system; Immundiagnostik, Germany). Stable growth rates of riboflavin level in the AHAC (with maximum values reached at 30-40 min) were observed for solutions D and R, regardless of the variant of corneal debridement. Moreover, throughout the whole follow-up period and regardless of the area of corneal debridement, the solution D provided a relatively lower concentration of riboflavin in the AHAC as compared to the two other solutions. At 30 minutes, when the cornea was considered ready for UV irradiation, the riboflavin level in the AHAC ranged from 385±26.1 μg/l (D-9) to 665±28 μg/l (R-9). In groups IR-9, IR-6, P-6, IR-3, and P-3 riboflavin levels were found to be in the same range starting at 20 minutes. However, even a sufficient concentration of riboflavin in the cornea or AHAC cannot guarantee safe and effective UV cross-linking, since the removed epithelium limits the area of the stroma that can be saturated with riboflavin, while the area of UV exposure is 8-10 mm. Safe and efficient standard UV cross-linking may be performed only under sufficient saturation of the

Characterization of Genipin-Modified Dentin Collagen

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Hiroko Nagaoka

2014-01-01

Full Text Available Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE, on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5% for several treatment times (0–24 h. Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P< 0.05. The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

Collagen macromolecular drug delivery systems

International Nuclear Information System (INIS)

Gilbert, D.L.

1988-01-01

The objective of this study was to examine collagen for use as a macromolecular drug delivery system by determining the mechanism of release through a matrix. Collagen membranes varying in porosity, crosslinking density, structure and crosslinker were fabricated. Collagen characterized by infrared spectroscopy and solution viscosity was determined to be pure and native. The collagen membranes were determined to possess native vs. non-native quaternary structure and porous vs. dense aggregate membranes by electron microscopy. Collagen monolithic devices containing a model macromolecule (inulin) were fabricated. In vitro release rates were found to be linear with respect to t 1/2 and were affected by crosslinking density, crosslinker and structure. The biodegradation of the collagen matrix was also examined. In vivo biocompatibility, degradation and 14 C-inulin release rates were evaluated subcutaneously in rats

Comparison of sequential vs same-day simultaneous collagen cross-linking and topography-guided PRK for treatment of keratoconus.

Science.gov (United States)

Kanellopoulos, Anastasios John

2009-09-01

The safety and efficacy of corneal collagen cross-linking (CXL) and topography-guided photorefractive keratectomy (PRK) using a different sequence and timing were evaluated in consecutive keratoconus cases. This study included a total of 325 eyes with keratoconus. Eyes were divided into two groups. The first group (n=127 eyes) underwent CXL with subsequent topography-guided PRK performed 6 months later (sequential group) and the second group (n=198 eyes) underwent CXL and PRK in a combined procedure on the same day (simultaneous group). Statistical differences were examined for pre- to postoperative changes in uncorrected (UCVA, logMAR) and best-spectacle-corrected visual acuity (BSCVA, logMAR), manifest refraction spherical equivalent (MRSE), keratometry (K), topography, central corneal thickness, endothelial cell count, corneal haze, and ectatic progression. Mean follow-up was 36+/-18 months (range: 24 to 68 months). At last follow-up in the sequential group, the mean UCVA improved from 0.9+/-0.3 logMAR to 0.49+/-0.25 logMAR, and mean BSCVA from 0.41+/-0.25 logMAR to 0.16+/-0.22 logMAR. Mean reduction in spherical equivalent refraction was 2.50+/-1.20 diopters (D), mean haze score was 1.2+/-0.5, and mean reduction in K was 2.75+/-1.30 D. In the simultaneous group, mean UCVA improved from 0.96+/-0.2 logMAR to 0.3+/-0.2 logMAR, and mean BSCVA from 0.39+/-0.3 logMAR to 0.11+/-0.16 logMAR. Mean reduction in spherical equivalent refraction was 3.20+/-1.40 D, mean haze score was 0.5+/-0.3, and mean reduction in K was 3.50+/-1.3 D. Endothelial cell count preoperatively and at last follow-up was unchanged (PPRK and CXL appears to be superior to sequential CXL with later PRK in the visual rehabilitation of progressing keratoconus. Copyright 2009, SLACK Incorporated.

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

Science.gov (United States)

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.

Intrastromal corneal ring segments for management of keratoconus

Directory of Open Access Journals (Sweden)

Sri Ganesh

2013-01-01

Full Text Available Keratoconus is a progressive corneal ectasia, which can be managed both by conservative measures like glasses or contact lenses in non-progressive cases or surgical procedures like collagen crosslinking (CXL with or without adjuvant measures like intrastromal corneal rings segments (ICRS or topography guided ablation. Various kinds of ICRS are available to the surgeon, but it is most essential to be able to plan the implantation of the ring to optimize outcomes. Aims: The aim of this study is to evaluate the visual outcome and progression in patients of keratoconus implanted with ICRS. Materials and Methods: Two different types of ICRS-Intacs (Addition Technology and Kerarings (Mediphacos Inc. were implanted in 2 different cohorts of patients and were followed-up to evaluate the outcome of the procedure. All patients underwent a complete ocular examination including best spectacle corrected visual acuity, slit lamp examination fundus examination, corneal topography and pachymetry. The ICRS implantation is done with CXL to stop the progression of the disease. Improvement in uncorrected visual acuity (UCVA, best spectacle corrected visual acuity and topographic changes were analyzed. Results: A significant improvement in keratometry and vision was seen in both groups. Conclusion: ICRS have been found to reduce corneal irregularity and flatten keratometry with improvement in UCVA and best corrected visual acuity.

Modification of mature non-reducible collagen cross-link concentrations in bovine m. gluteus medius and semitendinosus with steer age at slaughter, breed cross and growth promotants.

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Roy, B C; Sedgewick, G; Aalhus, J L; Basarab, J A; Bruce, H L

2015-12-01

Increased meat toughness with animal age has been attributed to mature trivalent collagen cross-link formation. Intramuscular trivalent collagen cross-link content may be decreased by reducing animal age at slaughter and/or inducing muscle re-modeling with growth promotants. This hypothesis was tested in m. gluteus medius (GM) and m. semitendinosus (ST) from 112 beef steers finished at either 12 to 13 (rapid growth) or 18 to 20 (slow growth) months of age. Hereford-Aberdeen Angus (HAA) or Charolais-Red Angus (CRA) steers were randomly assigned to receive implants (IMP), ractopamine (RAC), both IMP and RAC, or none (control). RAC decreased pyridinoline (mol/mol collagen) and IMP increased Ehrlich chromogen (EC) (mol/mol collagen) in the GM. In the ST, RAC increased EC (mol/mol collagen) but decreased EC (nmol/g raw muscle) in slow growing CRA steers. Also, IMP increased ST pyridinoline (nmol/g raw muscle) of slow-growing HAA steers. Results indicated alteration of perimysium collagen cross-links content in muscle in response to growth promotants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adjuvant corneal crosslinking to prevent hyperopic LASIK regression.

Science.gov (United States)

Aslanides, Ioannis M; Mukherjee, Achyut N

2013-01-01

To report the long term outcomes, safety, stability, and efficacy in a pilot series of simultaneous hyperopic laser assisted in situ keratomileusis (LASIK) and corneal crosslinking (CXL). A small cohort series of five eyes, with clinically suboptimal topography and/or thickness, underwent LASIK surgery with immediate riboflavin application under the flap, followed by UV light irradiation. Postoperative assessment was performed at 1, 3, 6, and 12 months, with late follow up at 4 years, and results were compared with a matched cohort that received LASIK only. The average age of the LASIK-CXL group was 39 years (26-46), and the average spherical equivalent hyperopic refractive error was +3.45 diopters (standard deviation 0.76; range 2.5 to 4.5). All eyes maintained refractive stability over the 4 years. There were no complications related to CXL, and topographic and clinical outcomes were as expected for standard LASIK. This limited series suggests that simultaneous LASIK and CXL for hyperopia is safe. Outcomes of the small cohort suggest that this technique may be promising for ameliorating hyperopic regression, presumed to be biomechanical in origin, and may also address ectasia risk.

Characterization of cross-linked porous gelatin carriers and their interaction with corneal endothelium: biopolymer concentration effect.

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Jui-Yang Lai

Full Text Available Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5-15 wt% on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 µm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young's modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5-10 wt%, the hydrogels have high freezable water content (0.89-0.93 and concentration of permeated glucose (591.3-615.5 µg/ml. These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer

Rate of riboflavin diffusion from intrastromal channels before corneal crosslinking.

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McQuaid, Rebecca; Mrochen, Michael; Vohnsen, Brian

2016-03-01

To determine the diffusion of riboflavin from intrastromal channels through the effective diffusion coefficients compared with traditional axial diffusion with epithelium on or off. Advanced Optical Imaging Laboratory, University College Dublin, and Wellington Eye Clinic, Sandyford, Dublin, Ireland. Experimental study. The rate of diffusion in whole-mounted porcine eyes was monitored for a 30 minutes using an optical setup with a charge-coupled device camera and a bandpass filter (central wavelength 550 nm and 40 nm bandpass) to image the fluorescence under ultraviolet illumination (365 nm wavelength). For comparison, an isotropic corneal stroma with an annular channel was modeled numerically for different diffusion constants and boundary conditions. Numerical and experimental results were compared, allowing determination of the effective diffusion coefficient for each case. Experimental results for 6 different riboflavin solutions were in all cases found to be higher than for the common crosslinking (CXL) riboflavin protocol, where the diffusion constant is D0 = 6.5 × 10(-5) mm(2)/sec. For the intrastromal channel, 2 isotonic solutions containing riboflavin 0.1% correlated with a diffusion constant of 5D0 = 32.5 × 10(-5) mm(2)/sec. Hypotonic solutions and transepithelium had a higher diffusion coefficient approaching 10D0 = 65.0 × 10(-5) mm(2)/sec, which is an order-of-magnitude increase compared with the typical diffusion coefficient found in standard CXL. In this study, riboflavin had a faster stromal diffusion when injected into a corneal channel than when applied as drops to the anterior corneal surface. Further numerical modeling might allow optimization of the channel structure for any specific choice of riboflavin. Copyright © 2016 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

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Sushovan Chowdhury

Full Text Available To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn.Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide (PLGA nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry.Pirfenidone prevented (P<0.05 increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05 reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn.Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases.

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

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Lanzini, Manuela; Curcio, Claudia; Spoerl, Eberhard; Calienno, Roberta; Mastropasqua, Alessandra; Colasante, Martina; Mastropasqua, Rodolfo; Nubile, Mario; Mastropasqua, Leonardo

2017-02-01

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm 2 UVA (group 4) and three for 9 min at 10 mW/cm 2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm 2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

EXPERIMENTAL REPAIR OF DEEP CORNEAL DEFECTS USING A BIO-CONSTRUCT COMPRISING A COLLAGEN TYPE I MATRIX LOADED WITH BUCCAL EPITHELIAL CELLS

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N. S. Egorova

2017-01-01

Full Text Available The  research  objective was  to study the  reparative effects of  the  collagen  type  I bio-construct loaded  with buccal epithelial cells, on the rabbit cornea after experimental keratectomy at various stages of treatment (on the 3rd, 7th, 14th, 3 0th days.Material  and methods.  The  experiments were  conducted on 20 rabbits  of  the  Chinchilla breed that  were  operated on cornea of both eyes aiming to inflict epithelial and stromal cornea defects. The collagen-based bio-construct bearing buccal epithelial cells was placed  over the cornea of the experimental eyes. The  cornea of the control  eyes was covered with smooth contact lens. After the surgery, a temporal blepharorrhaphy was performed and kept for 3 days. We studied macroand microscopic pattern of corneal regeneration at 3, 7, 14, and 30 days of experiment.Results. When  using the collaged-based bio-construct containing buccal epithelial cells, the complete epithelialization of the corneal defect occurred at mean 7 days earlier compared to that in the control eyes. Thus, the offered bio-construct stimulated the cell migration and proliferation at early stages of treatment (3–7 days reducing the inflammation activity.Conclusion. The bio-construct comprising a collagen type  I matrix loaded with buccal epithelial cells can provide an effective treatment option for corneal defects.

Simultaneous versus Sequential Accelerated Corneal Collagen Cross-Linking and Wave Front Guided PRK for Treatment of Keratoconus: Objective and Subjective Evaluation

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Waleed Ali Abou Samra

2016-01-01

Full Text Available Aim. To compare objective and subjective outcome after simultaneous wave front guided (WFG PRK and accelerated corneal cross-linking (CXL in patients with progressive keratoconus versus sequential WFG PRK 6 months after CXL. Methods. 62 eyes with progressive keratoconus were divided into two groups; the first including 30 eyes underwent simultaneous WFG PRK with accelerated CXL. The second including 32 eyes underwent subsequent WFG PRK performed 6 months later after accelerated CXL. Visual, refractive, topographic, and aberrometric data were determined preoperatively and during 1-year follow-up period and the results compared in between the 2 studied groups. Results. All evaluated visual, refractive, and aberrometric parameters demonstrated highly significant improvement in both studied groups (all P<0.001. A significant improvement was observed in keratometric and Q values. The improvement in all parameters was stable till the end of follow-up. Likewise, no significant difference was determined in between the 2 groups in any of recorded parameters. Subjective data revealed similarly significant improvement in both groups. Conclusions. WFG PRK and accelerated CXL is an effective and safe option to improve the vision in mild to moderate keratoconus. In one-year follow-up, there is no statistically significant difference between the simultaneous and sequential procedure.

Ultra B2 - Promoção de ligações covalentes do colágeno corneal (Corneal cross-linking no tratamento de ceratocone: resultados preliminares Corneal Cross-linking for the treatment of keratoconus: preliminary results

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Mirko R. Jankov II

2008-12-01

atingir diferença estatisticamente significante. Nenhum dos olhos perdeu linha de BSCVA, 12 mantiveram o BSCVA pré-operatório, 7 ganharam uma linha, 5 ganharam duas e 1 paciente ganhou três linhas de BSCVA. CONCLUSÕES: O tratamento com Ultra-B2 mostrou-se seguro (não apresentou perda de linha de visão corrigida e eficaz (manteve os parâmetros anatômicos e ópticos em estacionar a progressão da ectasia corneal. Houve redução, embora sem significância estatística, da curvatura corneal máxima, equivalente esférico e cilindro refrativo nos olhos com a córnea instável devida ao ceratocone.PURPOSE: To present early visual and keratometric results for corneal cross-linking with riboflavin and UV irradiation in patients with progressive keratoconus. METHODS: Twenty-five eyes of twenty patients (15 males and 5 females with a progressive keratoconus in the previous 6 months were followed. Unaided visual acuity (UVA, best spectacle corrected visual acuity (BSCVA, spherical equivalent (SEQ, manifest cylinder, and maximal corneal curvature (max K values were followed at 1, 3 and 6 months. All patients were submitted to corneal cross-linking using riboflavin (vitamin B2 as the photosensitizer and ultraviolet light (UV, wavelength 370 nm. Epithelium was removed with 20% alcohol, cornea was soaked with vitamin B2 for 15 min, and then irradiated with UV light for 30 min, after which a bandage contact lens (BCL was placed. RESULTS: UVA increased after one month (from 0.15 ± 0.15 to 0.23 ± 0.20, and went on increasing at 3 and 6 months, reaching statistical significance (p=0.025 e p=0.037, respectively. BSCVA increased from 0.41 ± 0.27 to 0.49 ± 0.29 at month six, without reaching statistical significance at any time point. Progression of keratoconus stopped in all patients, in contrast with progression in all of them in the six-month period prior to the surgery. Max K decreased by more than 2 D (from 53.02 ± 8.42 to 50.88 ± 6.05 D, SEQ less that 1 D (from -3.27 ± 4

Confocal Raman mapping of collagen cross-link and crystallinity of human dentin-enamel junction

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Slimani, Amel; Nouioua, Fares; Desoutter, Alban; Levallois, Bernard; Cuisinier, Frédéric J. G.; Tassery, Hervé; Terrer, Elodie; Salehi, Hamideh

2017-08-01

The separation zone between enamel and dentin [dentin-enamel junction (DEJ)] with different properties in biomechanical composition has an important role in preventing crack propagation from enamel to dentin. The understanding of the chemical structure (inorganic and organic components), physical properties, and chemical composition of the human DEJ could benefit biomimetic materials in dentistry. Spatial distribution of calcium phosphate crystallinity and the collagen crosslinks near DEJ were studied using confocal Raman microscopy and calculated by different methods. To obtain collagen crosslinking, the ratio of two peaks 1660 cm-1 over 1690 cm-1 (amide I bands) is calculated. For crystallinity, the inverse full-width at half maximum of phosphate peak at 960 cm-1, and the ratio of two Raman peaks of phosphate at 960/950 cm-1 is provided. In conclusion, the study of chemical and physical properties of DEJ provides many benefits in the biomaterial field to improve the synthesis of dental materials in respect to the natural properties of human teeth. Confocal Raman microscopy as a powerful tool provides the molecular structure to identify the changes along DEJ and can be expanded for other mineralized tissues.

Corneal Collagen Cross-Linking in the Management of Keratoconus in Canada: A Cost-Effectiveness Analysis.

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Leung, Victoria C; Pechlivanoglou, Petros; Chew, Hall F; Hatch, Wendy

2017-08-01

To use patient-level microsimulation models to evaluate the comparative cost-effectiveness of early corneal cross-linking (CXL) and conventional management with penetrating keratoplasty (PKP) when indicated in managing keratoconus in Canada. Cost-utility analysis using individual-based, state-transition microsimulation models. Simulated cohorts of 100 000 individuals with keratoconus who entered each treatment arm at 25 years of age. Fellow eyes were modeled separately. Simulated individuals lived up to a maximum of 110 years. We developed 2 state-transition microsimulation models to reflect the natural history of keratoconus progression and the impact of conventional management with PKP versus CXL. We collected data from the published literature to inform model parameters. We used realistic parameters that maximized the potential costs and complications of CXL, while minimizing those associated with PKP. In each treatment arm, we allowed simulated individuals to move through health states in monthly cycles from diagnosis until death. For each treatment strategy, we calculated the total cost and number of quality-adjusted life years (QALYs) gained. Costs were measured in Canadian dollars. Costs and QALYs were discounted at 5%, converting future costs and QALYs into present values. We used an incremental cost-effectiveness ratio (ICER = difference in lifetime costs/difference in lifetime health outcomes) to compare the cost-effectiveness of CXL versus conventional management with PKP. Lifetime costs and QALYs for CXL were estimated to be Can$5530 (Can$4512, discounted) and 50.12 QALYs (16.42 QALYs, discounted). Lifetime costs and QALYs for conventional management with PKP were Can$2675 (Can$1508, discounted) and 48.93 QALYs (16.09 QALYs, discounted). The discounted ICER comparing CXL to conventional management was Can$9090/QALY gained. Sensitivity analyses revealed that in general, parameter variations did not influence the cost-effectiveness of CXL. CXL is

Adjuvant corneal crosslinking to prevent hyperopic LASIK regression

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Aslanides IM

2013-03-01

Full Text Available Ioannis M Aslanides, Achyut N MukherjeeEmmetropia Mediterranean Eye Clinic, Heraklion, Crete, GreecePurpose: To report the long term outcomes, safety, stability, and efficacy in a pilot series of simultaneous hyperopic laser assisted in situ keratomileusis (LASIK and corneal crosslinking (CXL.Method: A small cohort series of five eyes, with clinically suboptimal topography and/or thickness, underwent LASIK surgery with immediate riboflavin application under the flap, followed by UV light irradiation. Postoperative assessment was performed at 1, 3, 6, and 12 months, with late follow up at 4 years, and results were compared with a matched cohort that received LASIK only.Results: The average age of the LASIK-CXL group was 39 years (26–46, and the average spherical equivalent hyperopic refractive error was +3.45 diopters (standard deviation 0.76; range 2.5 to 4.5. All eyes maintained refractive stability over the 4 years. There were no complications related to CXL, and topographic and clinical outcomes were as expected for standard LASIK.Conclusion: This limited series suggests that simultaneous LASIK and CXL for hyperopia is safe. Outcomes of the small cohort suggest that this technique may be promising for ameliorating hyperopic regression, presumed to be biomechanical in origin, and may also address ectasia risk.Keyword: CXL

Effect of pore size and cross-linking of a novel collagen-elastin dermal substitute on wound healing

NARCIS (Netherlands)

Boekema, B.K.H.L.; Vlig, M.; Damink, L.O.; Middelkoop, E.; Eummelen, L.; Buhren, A.V.; Ulrich, M.M.W.

2014-01-01

Collagen-elastin (CE) scaffolds are frequently used for dermal replacement in the treatment of full-thickness skin defects such as burn wounds. But little is known about the optimal pore size and level of cross-linking. Different formulations of dermal substitutes with unidirectional pores were

Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry.

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Matsuura, Takashi; Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu

2014-01-01

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Distinct Characteristics of Mandibular Bone Collagen Relative to Long Bone Collagen: Relevance to Clinical Dentistry

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Takashi Matsuura

2014-01-01

Full Text Available Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

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Barnes, Aileen M; Cabral, Wayne A; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C

2012-11-01

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. Published 2012 Wiley Periodicals, Inc.*This article is a US Government work and, as such, is in the public domain of the United States of America.

Topography-Guided PRK and Crosslinking in Eyes with Keratoconus and Post-LASIK Ectasia.

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Müller, T M; Lange, A P

2017-04-01

Topography-guided photorefractive keratectomy (TG-PRK) combined with corneal collagen crosslinking (CXL) has been shown to potentially improve vision and stabilize progression in patients with keratoconus (KC). We attempted to reproduce the previously published results using a different laser platform (AMARIS 500E) in patients with KC and post-LASIK ectasia (PLE). All of the 9 included eyes showed improved topography (Kmax, Kmean, RMS HOA, vertical coma, cylinder; p PRK and CXL may be a promising option to regularize and stabilize corneas with KC and PLE and improve visual acuity. Georg Thieme Verlag KG Stuttgart · New York.

In vivo argon laser vascular welding using thermal feedback: open and closed loop patency and collagen crosslinking

Energy Technology Data Exchange (ETDEWEB)

Small, W., LLNL

1997-02-28

An in vivo study of vascular welding with a fiber-delivered argon laser was conducted using a canine model. Longitudinal arteriotomies and venotomies were treated on femoral vein and artery. Laser energy was delivered to the vessel wall via a 400 {micro}m optical fiber. The surface temperature at the center of the laser spot was monitored in real time using a hollow glass optical fiber-based two-color infrared thermometer. The surface temperature was limited by either a room-temperature saline drip or direct feedback control of the laser using a mechanical shutter to alternately pass and block the laser. Acute patency was evaluated either visually (leak/no leak) or by in vivo burst pressure measurements. Biochemical assays were performed to investigate the possible laser-induced formation or destruction of enzymatically mediated covalent crosslinks between collagen molecules. Viable welds were created both with and without the use of feedback control. Tissues maintained at 50 C using feedback control had an elevated crosslink count compared to controls, while those irradiated without feedback control experienced a decrease. Differences between the volumetric heating associated with open and closed loop protocols may account for the different effects on collagen crosslinks. Covalent mechanisms may play a role in argon laser vascular fusion.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

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Anastasia, Luigi; Rota, Paola; Anastasia, Mario; Allevi, Pietro

2013-09-21

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Modificaciones en la estructura celular por microscopia confocal en pacientes con queratocono tratados por crosslinking Changes in the cell structure of patients with keratoconus under cross-linking treatment by using confocal microscopy

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Madelyn Jareño Ochoa

2012-12-01

corneal collagen crosslinking and confocal microscopy. The analyzed variables were the alterations in the sub-basal and sub-epithelial nervous plexuses, in the stromal nerves, the changes in keratocytes in the anterior and medium stroma, the changes of the endothelial cells, the depth of treatment as well as the presence and the progression of haze and the stromal edema. Results: there no sub-basal and sub-epithelial nervous plexuses during the first month, there were re-innervations after 6 months in 73,3 % of eyes and total recovery after 12 months. Likewise, there was no stromal nervous fibers in the first month, but after 6 months they were recovered in 96,7 % of patients and the total recovery was attained after a year. Rarefaction of the keratocytes in the anterior and the medium stroma was found during the first month in 96,7 % of cases, the onset of the cell repopulation was observed after 3 months in 83,3 % of cases and a total regeneration was evident after 1 year. The stromal edema was present in the first month in 83,3 % of cases, 6 months later was seen in 3.3 % and after 1 year, no patient was affected. Haze was identified on the first month and after 3 months in 100 % of patients. The haze grade II predominated in the first month, the grade I after 6 months and 60 % of patients presented with no haze after a year. Conclusions: the obtained results confirmed the criteria which establish that the corneal collagen crosslinking is a safe treatment method.

Corneal Cross-Linking Has No Effect on Matrix Metalloproteinase 9 and 13 Levels During Fungal Keratitis on the Early Stage.

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Kalkanci, Ayse; Bilgihan, Kamil; Ozdemir, Huseyin Baran; Yar Saglam, Atiye Seda; Karakurt, Funda; Erdogan, Merve

2018-04-01

The aim of our study was to investigate matrix metalloproteinases, MMP-9 and MMP-13 levels, in the rabbit model of Fusarium and Candida keratitis treated by corneal cross-linking (PACK-CXL). Rabbit corneas were inoculated with fungal inoculum for keratitis. Each group divided into four subgroups, including un-treated group, PACK-CXL group, voriconazole group and PACK-CXL plus voriconazole group. PACK-CXL was applied with 0.25% riboflavin in accelerated Dresden protocol, and 0.1% voriconazole drops were administered. All corneal buttons excised at tenth day after ophthalmological examination. Fungal cell counts and Scheiber scores were determined in all groups. Corneal tissue MMP mRNA levels were evaluated quantitative reverse transcriptase PCR. The difference in MMP-9 and MMP-13 levels at all groups was not statistically significant (p > 0.05). PACK-CXL with 0.25% riboflavin either alone or combined with antifungal drops was unable to provide decline in inflammatory findings in both macroscopic and microscopic levels similar to medical antifungal treatment.

Chitosan–Collagen Coated Magnetic Nanoparticles for Lipase Immobilization—New Type of “Enzyme Friendly” Polymer Shell Crosslinking with Squaric Acid

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Marta Ziegler-Borowska

2017-01-01

Full Text Available This article presents a novel route for crosslinking a polysaccharide and polysaccharide/protein shell coated on magnetic nanoparticles (MNPs surface via condensation reaction with squaric acid (SqA. The syntheses of four new types of collagen-, chitosan-, and chitosan–collagen coated magnetic nanoparticles as supports for enzyme immobilization have been done. Structure and morphology of prepared new materials were characterized by attenuated total reflectance Fourier-transform infrared (ATR-FTIR, XRD, and TEM analysis. Next, the immobilization of lipase from Candida rugosa was performed on the nanoparticles surface via N-(3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC/N-hydroxy-succinimide (NHS mechanism. The best results of lipase activity recovery and specific activities were observed for nanoparticles with polymer shell crosslinked via a novel procedure with squaric acid. The specific activity for lipase immobilized on materials crosslinked with SqA (52 U/mg lipase was about 2-fold higher than for enzyme immobilized on MNPs with glutaraldehyde addition (26 U/mg lipase. Moreover, a little hyperactivation of lipase immobilized on nanoparticles with SqA was observed (104% and 112%.

Determination of the relationship between collagen cross-links and the bone-tissue stiffness in the porcine mandibular condyle

NARCIS (Netherlands)

Willems, N.M.B.K.; Mulder, L.; Bank, R.A.; Grünheid, T.; Toonder, J.M.J. den; Zentner, A.; Langenbach, G.E.J.

2011-01-01

Although bone-tissue stiffness is closely related to the degree to which bone has been mineralized, other determinants are yet to be identified. We, therefore, examined the extent to which the mineralization degree, collagen, and its cross-links are related to bone-tissue stiffness. A total of 50

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

OpenAIRE

Reddy, G. Kesava

2004-01-01

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendo...

The Influence of Lamellar Orientation on Corneal Material Behavior: Biomechanical and Structural Changes in an Avian Corneal Disorder

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Elsheikh, Ahmed; Kassem, Wael; Kamma-Lorger, Christina S.; Hocking, Paul M.; White, Nick; Inglehearn, Chris F.; Ali, Manir; Meek, Keith M.

2011-01-01

Purpose. Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP). Methods. Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas. Results. RGE corneas displayed a 30–130% increase in material stiffness [Etangent(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; Etangent(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected. Conclusions. This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP. PMID:21051696

Tranexamic acid, an inhibitor of plasminogen activation, reduces urinary collagen cross-link excretion in both experimental and rheumatoid arthritis

NARCIS (Netherlands)

Ronday, H.K.; TeKoppele, J.M.; Greenwald, R.A.; Moak, S.A.; Roos, J.A.D.M. de; Dijkmans, B.A.C.; Breedveld, F.C.; Verheijen, J.H.

1998-01-01

The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and

Occlusal loading and cross-linking effects on dentin collagen degradation in physiological conditions.

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Turco, Gianluca; Frassetto, Andrea; Fontanive, Luca; Mazzoni, Annalisa; Cadenaro, Milena; Di Lenarda, Roberto; Tay, Franklin R; Pashley, David H; Breschi, Lorenzo

2016-02-01

This study evaluated the ability of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to improve the stability of demineralized dentin collagen matrices when subjected to mechanical cycling by means of Chewing Simulation (CS). Demineralized dentin disks were randomly assigned to four groups (N=4): (1) immersion in artificial saliva at 37°C for 30 days; (2) pre-treatment with 0.5 M EDC for 60 s, then stored as in Group 1; (3) CS challenge (50 N occlusal load, 30 s occlusal time plus 30 s with no load, for 30 days); (4) pre-treatment with 0.5 M EDC as in Group 2 and CS challenge as in Group 3. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptides. EDC treated specimens showed no significant telopeptides release, irrespective of the aging method. Cyclic stressing of EDC-untreated specimens caused significantly higher ICTP release at day 1, compared to static storage, while by days 3 and 4, the ICTP release in the cyclic group fell significantly below the static group, and then remained undetectable from 5 to 30 days. CTX release in the cyclic groups, on EDC-untreated control specimens was always lower than in the static group in days 1-4, and then fell to undetectable for 30 days. This study showed that chewing stresses applied to control untreated demineralized dentin increased degradation of collagen in terms of CTX release, while collagen crosslinking agents may prevent dentin collagen degradation, irrespective of simulated occlusal function. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Corneal biomechanical properties from air-puff corneal deformation imaging

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Marcos, Susana; Kling, Sabine; Bekesi, Nandor; Dorronsoro, Carlos

2014-02-01

The combination of air-puff systems with real-time corneal imaging (i.e. Optical Coherence Tomography (OCT), or Scheimpflug) is a promising approach to assess the dynamic biomechanical properties of the corneal tissue in vivo. In this study we present an experimental system which, together with finite element modeling, allows measurements of corneal biomechanical properties from corneal deformation imaging, both ex vivo and in vivo. A spectral OCT instrument combined with an air puff from a non-contact tonometer in a non-collinear configuration was used to image the corneal deformation over full corneal cross-sections, as well as to obtain high speed measurements of the temporal deformation of the corneal apex. Quantitative analysis allows direct extraction of several deformation parameters, such as apex indentation across time, maximal indentation depth, temporal symmetry and peak distance at maximal deformation. The potential of the technique is demonstrated and compared to air-puff imaging with Scheimpflug. Measurements ex vivo were performed on 14 freshly enucleated porcine eyes and five human donor eyes. Measurements in vivo were performed on nine human eyes. Corneal deformation was studied as a function of Intraocular Pressure (IOP, 15-45 mmHg), dehydration, changes in corneal rigidity (produced by UV corneal cross-linking, CXL), and different boundary conditions (sclera, ocular muscles). Geometrical deformation parameters were used as input for inverse finite element simulation to retrieve the corneal dynamic elastic and viscoelastic parameters. Temporal and spatial deformation profiles were very sensitive to the IOP. CXL produced a significant reduction of the cornea indentation (1.41x), and a change in the temporal symmetry of the corneal deformation profile (1.65x), indicating a change in the viscoelastic properties with treatment. Combining air-puff with dynamic imaging and finite element modeling allows characterizing the corneal biomechanics in-vivo.

Comparison of the Early Clinical Outcomes between Combined Small-Incision Lenticule Extraction and Collagen Cross-Linking versus SMILE for Myopia

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Alex L. K. Ng

2016-01-01

Full Text Available Background. To compare the early outcome of combined SMILE and collagen crosslinking (SMILE Xtra with SMILE. Method. Prospective, comparative interventional study of 21 eyes receiving SMILE Xtra using a low energy protocol and 32 control eyes receiving SMILE only. The outcomes were compared at 1, 3, and 6 months postoperatively. Results. Both groups had myopia with spherical equivalent refraction (SEQ > 4.00 D. The SMILE Xtra group had thinner preoperative central corneal thickness and residual stromal bed thickness (p<0.021. At 6 months, no eyes lost more than 1 line in corrected distance visual acuity. The safety index was 0.96±0.06 and 1.00±0.00 in SMILE Xtra and control, respectively (p<0.001. 89% and 94% of eyes were within ±0.50 D of target refraction, respectively, with the mean error in SEQ correction being -0.17±0.26 D for SMILE Xtra and +0.03±0.25 D for control (p=0.021. The efficacy index was 0.88±0.13 and 0.97±0.06, respectively (p=0.005. Conclusion. SMILE Xtra had good overall safety profile and predictability at 6 months. However, when compared with control, the safety index and efficacy index were statistically significantly lower in the early postoperative period.

A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration.

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Gore, Daniel M; O'Brart, David P; French, Paul; Dunsby, Chris; Allan, Bruce D

2015-12-01

To measure corneal riboflavin penetration using different transepithelial iontophoresis protocols. Freshly enucleated rabbit eyes were divided into nine treatment groups of 4 eyes. One group, in which 0.1% wt/vol riboflavin was applied for 30 minutes without iontophoresis after corneal epithelial debridement, acted as a control. The remaining groups were treated with an intact epithelium using different riboflavin formulations and varying iontophoresis current, soak, and rinse times. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35 μm thick were then imaged immediately by two-photon fluorescence microscopy, using image processing software to quantify stromal riboflavin concentration at different corneal depths. In the epithelium-on iontophoresis treatment groups, greater stromal riboflavin penetration was achieved with higher-concentration riboflavin solutions, greater iontophoresis dosage, and longer solution contact times. A protocol utilizing 0.25% wt/vol riboflavin with benzalkonium chloride (BAC) 0.01% and two cycles of applied current and subsequent soaking (1 mA 5 minutes, soak 5 minutes; 0.5 mA 5 minutes, soak 5 minutes) achieved similar stromal riboflavin penetration to epithelium-off controls. The best-performing non-BAC-containing protocol produced stromal riboflavin penetration approximately 60% that of epithelium-off controls. Riboflavin solutions containing saline resulted in minimal stromal penetration. Riboflavin loading within the epithelium was equivalent to or higher than that in the subjacent stroma, despite rinsing the ocular surface with balanced salt solution. Modified iontophoresis protocols can significantly improve transepithelial riboflavin penetration in experimental corneal collagen cross-linking.

The comparative safety of genipin versus UVA-riboflavin crosslinking of rabbit corneas

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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

New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging.

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

2015-09-01

To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti:Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.

Enhancing amine terminals in an amine-deprived collagen matrix.

LENUS (Irish Health Repository)

Tiong, William H C

2008-10-21

Collagen, though widely used as a core biomaterial in many clinical applications, is often limited by its rapid degradability which prevents full exploitation of its potential in vivo. Polyamidoamine (PAMAM) dendrimer, a highly branched macromolecule, possesses versatile multiterminal amine surface groups that enable them to be tethered to collagen molecules and enhance their potential. In this study, we hypothesized that incorporation of PAMAM dendrimer in a collagen matrix through cross-linking will result in a durable, cross-linked collagen biomaterial with free -NH 2 groups available for further multi-biomolecular tethering. The aim of this study was to assess the physicochemical properties of a G1 PAMAM cross-linked collagen matrix and its cellular sustainability in vitro. Different amounts of G1 PAMAM dendrimer (5 or 10 mg) were integrated into bovine-derived collagen matrices through a cross-linking process, mediated by 5 or 25 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 5 mM N-hydroxysuccinimide (NHS) and 50 mM 2-morpholinoethane sulfonic acid buffer at pH 5.5. The physicochemical properties of resultant matrices were investigated with scanning electron microscopy (SEM), collagenase degradation assay, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectra, and ninhydrin assay. Cellular sustainability of the matrices was assessed with Alamar Blue assay and SEM. There was no significant difference in cellular behavior between the treated and nontreated groups. However, the benefit of incorporating PAMAM in the cross-linking reaction was limited when higher concentrations of either agent were used. These results confirm the hypothesis that PAMAM dendrimer can be incorporated in the collagen cross-linking process in order to modulate the properties of the resulting cross-linked collagen biomaterial with free -NH 2 groups available for multi-biomolecular tethering.

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions.

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Mitra, Tapas; Sailakshmi, G; Gnanamani, A; Mandal, A B

2012-05-01

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young's modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.

Fabrication and in vitro evaluation of the collagen/hyaluronic acid PEM coating crosslinked with functionalized RGD peptide on titanium.

Science.gov (United States)

Huang, Ying; Luo, Qiaojie; Li, Xiaodong; Zhang, Feng; Zhao, Shifang

2012-02-01

Surface modification of titanium (Ti) using biomolecules has attracted much attention recently. In this study, a new strategy has been employed to construct a stable and bioactive coating on Ti. To this end, a derivative of hyaluronic acid (HA), i.e. HA-GRGDSPC-(SH), was synthesized. The disulfide-crosslinked Arg-Gly-Asp (RGD)-containing collagen/hyaluronic acid polyelectrolyte membrane (PEM) coating was then fabricated on Ti through the alternate deposition of collagen and HA-GRGDSPC-(SH) with five assembly cycles and subsequent crosslinking via converting free sulphydryl groups into disulfide linkages (RGD-CHC-Ti group). The assembly processes for PEM coating and the physicochemical properties of the coating were carefully characterized. The stability of PEM coating in phosphate-buffered saline solution could be adjusted by the crosslinking degree, while its degradation behaviors in the presence of glutathione were glutathione concentration dependent. The adhesion and proliferation of MC3T3-E1 cells were significantly enhanced in the RGD-CHC-Ti group. Up-regulated bone specific genes, enhanced alkaline phosphatase activity and osteocalcin production, the increased areas of mineralization were also observed in the RGD-CHC-Ti group. These results indicate that the strategy employed herein may function as an effective way to construct stable, RGD-containing bioactive coatings on Ti. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effect of pore size and cross-linking of a novel collagen-elastin dermal substitute on wound healing.

Science.gov (United States)

Boekema, Bouke K H L; Vlig, Marcel; Olde Damink, Leon; Middelkoop, Esther; Eummelen, Lizette; Bühren, Anne V; Ulrich, Magda M W

2014-02-01

Collagen-elastin (CE) scaffolds are frequently used for dermal replacement in the treatment of full-thickness skin defects such as burn wounds. But little is known about the optimal pore size and level of cross-linking. Different formulations of dermal substitutes with unidirectional pores were tested in porcine full-thickness wounds in combination with autologous split skin mesh grafts (SSG). Effect on wound healing was evaluated both macro- and microscopically. CE scaffolds with a pore size of 80 or 100 μm resulted in good wound healing after one-stage grafting. Application of scaffolds with a larger average pore size (120 μm) resulted in more myofibroblasts and more foreign body giant cells (FBGC). Moderate crosslinking impaired wound healing as it resulted in more wound contraction, more FBGC and increased epidermal thickness compared to no cross-linking. In addition, take rate and redness were negatively affected compared to SSG only. Vascularization and the number of myofibroblasts were not affected by cross-linking. Surprisingly, stability of cross-linked scaffolds was not increased in the wound environment, in contrast to in vitro results. Cross-linking reduced the proliferation of fibroblasts in vitro, which might explain the reduced clinical outcome. The non-cross-linked CE substitute with unidirectional pores allowed one-stage grafting of SSG, resulting in good wound healing. In addition, only a very mild foreign body reaction was observed. Cross-linking of CE scaffolds negatively affected wound healing on several important parameters. The optimal non-cross-linked CE substitute is a promising candidate for future clinical evaluation.

Large proteoglycan complexes and disturbed collagen architecture in the corneal extracellular matrix of mucopolysaccharidosis type VII (Sly syndrome).

Science.gov (United States)

Young, Robert D; Liskova, Petra; Pinali, Christian; Palka, Barbara P; Palos, Michalis; Jirsova, Katerina; Hrdlickova, Enkela; Tesarova, Marketa; Elleder, Milan; Zeman, Jiri; Meek, Keith M; Knupp, Carlo; Quantock, Andrew J

2011-08-24

Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.

Epithelium-on photorefractive intrastromal cross-linking (PiXL for reduction of low myopia

Directory of Open Access Journals (Sweden)

Lim WK

2017-06-01

Full Text Available Wee Kiak Lim,1,2 Zhi Da Soh,1 Harold Kah Yen Choi,1 Julian Thiam Siew Theng1,3 1Eagle Eye Centre, Mount Alvernia Hospital, 2Department of Ophthalmology, Tan Tock Seng Hospital, 3Department of Ophthalmology, Khoo Teck Puat Hospital, Singapore Purpose: To report the 9–12-month outcomes of a novel procedure for reduction of low myopia through epithelium-on photorefractive intrastromal cross-linking (PiXL with customized control of topographic distribution of ultraviolet (UV-fluence. Method: Myopic patients with normal (non-ectatic corneas underwent the PiXL procedure for reduction of low myopia. PiXL treatments were delivered through selective application of UVA light based on the refractive error of each patient. Clinical evaluation included safety (corrected distance visual acuity, endothelial cell count, central corneal thickness, anterior ocular health and efficacy (uncorrected distance visual acuity, manifest refraction, K-mean examinations. In addition, a patient satisfaction survey was conducted at 9 months post-procedure to evaluate patients’ subjective experience with the procedure. Results: Fourteen myopic eyes (mean manifest refraction spherical equivalent –1.62±0.6D; range –0.75 to –2.65D of 8 subjects (mean age 30 years old; range 24–51 years old were enrolled in the study. At 12 months post-procedure, a mean manifest refraction spherical equivalent reduction of 0.72±0.43D (P<0.001 was observed, with a corresponding gain in uncorrected visual acuity of 0.25 logMAR and mean K-mean flattening of 0.47±0.46D. All patients achieved best corrected visual acuity of 20/20 or better from 1 month onward. There were no cases of ocular infection or secondary changes to the crystalline lens and retina due to UV exposure, while transient corneal haze subsided gradually. Conclusion: The epithelium-on PiXL procedure was safe and effective in reducing myopic refractive error in this study with up to 12 months follow-up. Early results of

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

Science.gov (United States)

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.

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.

Intracorneal ring segments implantation followed by same-day topography-guided PRK and corneal collagen CXL in low to moderate keratoconus.

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Al-Tuwairqi, Waleed; Sinjab, Mazen M

2013-01-01

To evaluate the safety and efficacy of intrastromal corneal ring segments (ICRS) implantation followed by same-day topography-guided photorefractive keratectomy (PRK) and ultraviolet-A/riboflavin collagen cross-linking (CXL) in patients with low to moderate keratoconus. Patients with low to moderate keratoconus and contact lens intolerance were included in the study. All patients first underwent femtosecond laser-enabled placement of ICRS (Keraring, Mediphacos) (first step). Same-day topography-guided PRK and CXL (second step) were subsequently performed in all patients after the refraction was stable (average 6 months [range: 3 to 11 months]). Thirteen eyes from 13 patients were included in the study. Based on values before the first step and 6 months after the second step, significant improvements were noted in uncorrected distance visual acuity (0.7±0.32 logMAR vs 0.08±0.08 logMAR), corrected distance visual acuity (CDVA) (0.16±0.19 logMAR vs 0.02±0.04 log-MAR), sphere (-3.65±3.08 diopters [D] vs 0.06±1.6 D), astigmatism (-3.31±1.5 D vs -0.98±0.75 D), average K (47.28±1.99 D vs 41.42±3.22 D), and coma (2.36±1.23 μm vs 1.47±0.68 μm) (PPRK/CXL may be a reasonable option for improving visual acuity in patients with low to moderate keratoconus. Copyright 2013, SLACK Incorporated.

Microfluidic production of bioactive fibrin micro-beads embedded in crosslinked collagen used as an injectable bulking agent for urinary incontinence treatment.

Science.gov (United States)

Vardar, E; Larsson, H M; Allazetta, S; Engelhardt, E M; Pinnagoda, K; Vythilingam, G; Hubbell, J A; Lutolf, M P; Frey, P

2018-02-01

Endoscopic injection of bulking agents has been widely used to treat urinary incontinence, often due to urethral sphincter complex insufficiency. The aim of the study was to develop a novel injectable bioactive collagen-fibrin bulking agent restoring long-term continence by functional muscle tissue regeneration. Fibrin micro-beads were engineered using a droplet microfluidic system. They had an average diameter of 140 μm and recombinant fibrin-binding insulin-like growth factor-1 (α 2 PI 1-8 -MMP-IGF-1) was covalently conjugated to the beads. A plasmin fibrin degradation assay showed that 72.5% of the initial amount of α 2 PI 1-8 -MMP-IGF-1 loaded into the micro-beads was retained within the fibrin micro-beads. In vitro, the growth factor modified fibrin micro-beads enhanced cell attachment and the migration of human urinary tract smooth muscle cells, however, no change of the cellular metabolic activity was seen. These bioactive micro-beads were mixed with genipin-crosslinked homogenized collagen, acting as a carrier. The collagen concentration, the degree of crosslinking, and the mechanical behavior of this bioactive collagen-fibrin injectable were comparable to reference samples. This novel injectable showed no burst release of the growth factor, had a positive effect on cell behavior and may therefore induce smooth muscle regeneration in vivo, necessary for the functional treatment of stress and other urinary incontinences. Urinary incontinence is involuntary urine leakage, resulting from a deficient function of the sphincter muscle complex. Yet there is no functional cure for this devastating condition using current treatment options. Applied physical and surgical therapies have limited success. In this study, a novel bioactive injectable bulking agent, triggering new muscle regeneration at the injection site, has been evaluated. This injectable consists of cross-linked collagen and fibrin micro-beads, functionalized with bound insulin-like growth factor

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

Science.gov (United States)

Krishnakumar, Gopal Shankar; Gostynska, Natalia; Campodoni, Elisabetta; Dapporto, Massimiliano; Montesi, Monica; Panseri, Silvia; Tampieri, Anna; Kon, Elizaveta; Marcacci, Maurilio; Sprio, Simone; Sandri, Monica

2017-08-01

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Fabrication of Collagen Gel Hollow Fibers by Covalent Cross-Linking for Construction of Bioengineering Renal Tubules.

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Shen, Chong; Zhang, Guoliang; Wang, Qichen; Meng, Qin

2015-09-09

Collagen, the most used natural biomacromolecule, has been extensively utilized to make scaffolds for cell cultures in tissue engineering, but has never been fabricated into the configuration of a hollow fiber (HF) for cell culture due to its poor mechanical properties. In this study, renal tubular cell-laden collagen hollow fiber (Col HF) was fabricated by dissolving sacrificial Ca-alginate cores from collagen shells strengthened by carbodiimide cross-linking. The inner/outer diameters of the Col HF were precisely controlled by the flow rates of core alginate/shell collagen solution in the microfluidic device. As found, the renal tubular cells self-assembled into renal tubules with diameters of 50-200 μm post to the culture in Col HF for 10 days. According to the 3D reconstructed confocal images or HE staining, the renal cells appeared as a tight tubular monolayer on the Col HF inner surface, sustaining more 3D cell morphology than the cell layer on the 2D flat collagen gel surface. Moreover, compared with the cultures in either a Transwell or polymer HF membrane, the renal tubules in Col HF exhibited at least 1-fold higher activity on brush border enzymes of alkaline phosphatase and γ-glutamyltransferase, consistent with their gene expressions. The enhancement occurred similarly on multidrug resistance protein 2 and glucose uptake. Such bioengineered renal tubules in Col HF will present great potential as alternatives to synthetic HF in both clinical use and pharmaceutical investigation.

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.

The spectra character of photodegraded the pyridinoline cross-links by Hypocrellin B

International Nuclear Information System (INIS)

Zhang Jucheng; Chen Rui; Liu Wei; Chen Zhuo; Shu Lidan; Liu Yingji

2011-01-01

Pyridinoline cross-links is one of the cross-link formation in collagen which in cell matrix, many research shown that this cross-link cause the fibrosis. Hypocrellin B (HB) is one of the nature photosensitizers, this work investigated the pyridinoline cross-link in collagen was photodegraded by HB. The result shown HB can degrade the pyridinoline cross-link with photo. This is to say, HB may be use as the photodynamic reagent to study the fibrosis.

Rate of Corneal Collagen Crosslinking Redo in Private Practice: Risk Factors and Safety

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Joelle Antoun

2015-01-01

Full Text Available Objective. To report the rate of progression of keratectasia after primary crosslinking (CXL and evaluate the safety and efficiency of CXL redo. Materials and Methods. We conducted a retrospective analysis of the patients who underwent CXL between 2010 and 2013 at the Beirut Eye Specialist Hospital, Lebanon. Progression of keratectasia was based on the presence of an increase in maximum keratometry of 1.00 D, a change in the map difference between two consecutive topographies of 1.00 D, a deterioration of visual acuity, or any change in the refraction. Primary and redo CXL were done using the same protocol. Results. Among the 221 eyes of 130 patients who underwent CXL, 7 eyes (3.17% of five patients met the criteria of progression. All patients reported a history of allergic conjunctivitis and eye rubbing and progressed within 9 to 48 months. No complications were noted and all patients were stable 1 year after CXL redo. Conclusion. Allergic conjunctivitis and eye rubbing were the only risk factors associated with keratoconus progression after CXL. A close followup is thus mandatory, even years after the procedure. CXL redo seems to be a safe and efficient technique to halt the progression after a primary CXL.

Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

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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.

A Comparative Study of Vertebrate Corneal Structure: The Evolution of a Refractive Lens.

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Winkler, Moritz; Shoa, Golroxan; Tran, Stephanie T; Xie, Yilu; Thomasy, Sarah; Raghunathan, Vijay K; Murphy, Christopher; Brown, Donald J; Jester, James V

2015-04-01

Although corneal curvature plays an important role in determining the refractive power of the vertebrate eye, the mechanisms controlling corneal shape remain largely unknown. To address this question, we performed a comparative study of vertebrate corneal structure to identify potential evolutionarily based changes that correlate with the development of a corneal refractive lens. Nonlinear optical (NLO) imaging of second-harmonic-generated (SHG) signals was used to image collagen and three-dimensionally reconstruct the lamellar organization in corneas from different vertebrate clades. Second-harmonic-generated images taken normal to the corneal surface showed that corneal collagen in all nonmammalian vertebrates was organized into sheets (fish and amphibians) or ribbons (reptiles and birds) extending from limbus to limbus that were oriented nearly orthogonal (ranging from 77.7°-88.2°) to their neighbors. The slight angular offset (2°-13°) created a rotational pattern that continued throughout the full thickness in fish and amphibians and to the very posterior layers in reptiles and birds. Interactions between lamellae were limited to "sutural" fibers in cartilaginous fish, and occasional lamellar branching in fish and amphibians. There was a marked increase in lamellar branching in higher vertebrates, such that birds ≫ reptiles > amphibians > fish. By contrast, mammalian corneas showed a nearly random collagen fiber organization with no orthogonal, chiral pattern. Our data indicate that nonmammalian vertebrate corneas share a common orthogonal collagen structural organization that shows increased lamellar branching in higher vertebrate species. Importantly, mammalian corneas showed a different structural organization, suggesting a divergent evolutionary background.

Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds

Science.gov (United States)

Boote, Craig; Du, Yiqin; Morgan, Sian; Harris, Jonathan; Kamma-Lorger, Christina S.; Hayes, Sally; Lathrop, Kira L.; Roh, Danny S.; Burrow, Michael K.; Hiller, Jennifer; Terrill, Nicholas J.; Funderburgh, James L.; Meek, Keith M.

2012-01-01

Purpose. The mouse has become an important wound healing model with which to study corneal fibrosis, a frequent complication of refractive surgery. The aim of the current study was to quantify changes in stromal ultrastructure and light scatter that characterize fibrosis in mouse corneal debridement wounds. Methods. Epithelial debridement wounds, with and without removal of basement membrane, were produced in C57BL/6 mice. Corneal opacity was measured using optical coherence tomography, and collagen diameter and matrix order were quantified by x-ray scattering. Electron microscopy was used to visualize proteoglycans. Quantitative PCR (Q-PCR) measured mRNA transcript levels for several quiescent and fibrotic markers. Results. Epithelial debridement without basement membrane disruption produced a significant increase in matrix disorder at 8 weeks, but minimal corneal opacity. In contrast, basement membrane penetration led to increases in light scatter, matrix disorder, and collagen diameter, accompanied by the appearance of abnormally large proteoglycans in the subepithelial stroma. This group also demonstrated upregulation of several quiescent and fibrotic markers 2 to 4 weeks after wounding. Conclusions. Fibrotic corneal wound healing in mice involves extensive changes to collagen and proteoglycan ultrastructure, consistent with deposition of opaque scar tissue. Epithelial basement membrane penetration is a deciding factor determining the degree of ultrastructural changes and resulting opacity. PMID:22467580

Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes - Relevance of glycated collagen products versus HbA(1c) as markers of diabetic complications

NARCIS (Netherlands)

Monnier, VM; Bautista, O; Kenny, D; Sell, DR; Fogarty, J; Dahms, W; Cleary, PA; Lachin, J; Genuth, S

The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N-epsilon-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the

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.

Late Stage of Corneal Decompensation Caused by Progressive Keratoconus: Can We Treat It and Save the Cornea?

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Igor Knezović

2015-01-01

Full Text Available Aim. To report a case of 40-year-old male with progressive bilateral keratoconus who had undergone transepithelial phototherapeutic keratectomy (TE-PTK and corneal collagen cross-linking (CXL using hypoosmolar riboflavin solution in a same day procedure. Methods. Eye examination showed that UCDVA on both eyes was 0,01 according to Snellen charts, and slit lamp biomicroscopy showed paracentral diffuse intrastromal corneal haze. Anterior OCT marked stromal hyperreflective zones and localized paracentral thinning of the cornea. Scheimpflug tomography noted keratoconus stages III-IV on both eyes. After 40/35 microns TE-PTK, a CXL was performed for 30 minutes using hypoosmolar riboflavin solution. The left eye was treated first and the right eye 1 month after. Follow-up period was 10 months. Results. One month after the treatment both eyes showed improvement in corneal topography and the UCDVA was better. Eight months after the treatment BSCVA improved to 0,6 in both eyes using Rose K2 contact lenses and remained stable. Conclusion. TE-PTK and CXL using hypotonic riboflavin solution as a same day procedure have been shown to be a safe and promising method in this case of progressive keratoconus. It was necessary to consider certain parameters that could influence the safety and the final outcome of this combined protocol.

Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: experimentally supported micromechanical explanation of bone strength.

Science.gov (United States)

Fritsch, Andreas; Hellmich, Christian; Dormieux, Luc

2009-09-21

There is an ongoing discussion on how bone strength could be explained from its internal structure and composition. Reviewing recent experimental and molecular dynamics studies, we here propose a new vision on bone material failure: mutual ductile sliding of hydroxyapatite mineral crystals along layered water films is followed by rupture of collagen crosslinks. In order to cast this vision into a mathematical form, a multiscale continuum micromechanics theory for upscaling of elastoplastic properties is developed, based on the concept of concentration and influence tensors for eigenstressed microheterogeneous materials. The model reflects bone's hierarchical organization, in terms of representative volume elements for cortical bone, for extravascular and extracellular bone material, for mineralized fibrils and the extrafibrillar space, and for wet collagen. In order to get access to the stress states at the interfaces between crystals, the extrafibrillar mineral is resolved into an infinite amount of cylindrical material phases oriented in all directions in space. The multiscale micromechanics model is shown to be able to satisfactorily predict the strength characteristics of different bones from different species, on the basis of their mineral/collagen content, their intercrystalline, intermolecular, lacunar, and vascular porosities, and the elastic and strength properties of hydroxyapatite and (molecular) collagen.

Corneal-Wavefront guided transepithelial photorefractive keratectomy after corneal collagen cross linking in keratoconus

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Massimo Camellin

2017-01-01

Conclusions: Corneal-Wavefront guided transepithelial PRK ablation profiles after conventional CXL yields to good visual, optical, and refractive results. These treatments are safe and efficacious for the correction of refracto-therapeutic problems in keratoconic patients.

Tratamento aditivo do ceratocone por 'crosslinking' do colágeno após implante de anel de Ferrara Addictive treatment of keratoconus with collagen crosslinking after Ferrara ring implant

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Edna Almodin

2009-06-01

Full Text Available OBJETIVO: Este trabalho tem como objetivo relatar experiência com utilização do crosslinking após implante de anel intracorneano de Ferrara (Ferrara Ophtalmic visando a melhora refrativa e estabilização corneana. MÉTODOS: Seis pacientes com idade entre 26 e 38 anos foram submetidos ao tratamento com crosslinking após implante de anel de Ferrara. Cinco pacientes receberam 2 segmentos de anel e um paciente recebeu um segmento. Depois de 4 a 55 meses os pacientes foram submetidos a aplicação do "crosslinking" com ultra-violeta de acordo com o protocolo estabelecido por Seiler & cols. RESULTADOS: Quatro olhos apresentaram alteração progressiva da refração e 2 apresentaram refração sem alteração progressiva. A topografia mostrou queda acentuada da curvatura e o estudo da lâmpada de fenda mostrou anel em boa profundidade durante um ano de pós-operatório em 100% dos olhos. A microscopia especular não mostrou alteração significativa e a acuidade visual se mostrou semelhante ao pré-operatório. CONCLUSÃO: O crosslinking poderá ajudar a manter ceratocones estáveis, não-somente nos casos de progressão, mas também nos cones evolutivos com implante de anel.PURPOSE: To describe our experience with crosslinking after Ferrara ring implant for better vision and corneal stabilization. METHODS: six patients with age of 26 - 38 years old had treatment with crosslinking after Ferrara ring implant. Five patients had 2 ring segments implanted and one patient had one segment implanted. After 4 to 55 months the patients were submitted to crosslinking with UVA light according to Seiler et al protocol. RESULTS: Four eyes had progressive alteration of refraction and 2 patients had no alteration of refraction. The topography showed flattener curvature and at the slip lamp 100% of the eyes had good depth after one year post operatively. The specular microscopy didn't show any significant alteration and the visual acuity was similar before the

Transepithelial photorefractive keratectomy with crosslinking for keratoconus.

Science.gov (United States)

Mukherjee, Achyut N; Selimis, Vasilis; Aslanides, Ioannis

2013-01-01

To analyse visual, refractive and topographic outcomes of combining transepithelial photorefractive keratectomy (tPRK) with simultaneous corneal crosslinking for the visual rehabilitation of contact lens intolerant keratoconus patients. Patients with topographically significant keratoconus, limited corrected vision and intolerant of contact lenses were prospectively recruited, subject to ethical approval and consent. All patients underwent single step aspheric tPRK and sequential crosslinking. Preoperative vision, refraction, corneal topography and wavefront were assessed, with postoperative assessment at 1, 3, 6, and 12 months. 22 eyes of 14 patients were included in the pilot study. Mean age was 32 years (SD 6.8, range 24 to 43). Mean preoperative unaided vision was 1.39 LogMAR (SD 0.5) best corrected 0.31 LogMAR (SD 0.2). Mean preoperative spherical equivalent was -2.74 Diopters (D) (SD 4.1 range -12.25 to +7.75), and mean cylinder -2.9 D (SD 1.2, range 0 to -5.5). Mean central corneal thickness was 461um (SD 29, range 411 to 516). Vision improved postoperatively; unaided 0.32 LogMAR (SD 0.4), best corrected 0.11 (SD 0.13) (P=<0.005). Mean postoperative cylinder was -1.4D (SD1.2), significantly reduced (p<0.005). Maximum keratometry (Kmax) was stable throughout postoperative follow up. (p<0.05). Non topographic transepithelial PRK with simultaneous crosslinking improves vision, and may offer an alternative to keratoplasty in contact lens intolerant keratoconus. Further comparative studies to topographic PRK techniques are indicated.

Chemical Stabilisation of Collagen as a Biomimetic

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R. Gordon Paul

2003-01-01

Full Text Available Collagen is the most abundant protein in animals and because of its high mechanical strength and good resistance to degradation has been utilized in a wide range of products in industry whilst its low antigenicity has resulted in its widespread use in medicine. Collagen products can be purified from fibres, molecules reconstituted as fibres or from specific recombinant polypeptides with preferred properties. A common feature of all these biomaterials is the need for stable chemical cross-linking to control the mechanical properties and the residence time in the body, and to some extent the immunogenicity of the device. This can be achieved by a number of different cross-linking agents that react with specific amino acid residues on the collagen molecule imparting individual biochemical, thermal and mechanical characteristics to the biomaterial. In this review we have summarised the major techniques for testing these characteristics and the mechanisms involved in the variety of cross-linking reactions to achieve particular properties..

Porcine skin as a source of biodegradable matrices: alkaline treatment and glutaraldehyde crosslinking

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Fabiana T. Rodrigues

2010-06-01

Full Text Available In this work, the modifications promoted by alkaline hydrolysis and glutaraldehyde (GA crosslinking on type I collagen found in porcine skin have been studied. Collagen matrices were obtained from the alkaline hydrolysis of porcine skin, with subsequent GA crosslinking in different concentrations and reaction times. The elastin content determination showed that independent of the treatment, elastin was present in the matrices. Results obtained from in vitro trypsin degradation indicated that with the increase of GA concentration and reaction time, the degradation rate decreased. From thermogravimetry and differential scanning calorimetry analysis it can be observed that the collagen in the matrices becomes more resistant to thermal degradation as a consequence of the increasing crosslink degree. Scanning electron microscopy analysis indicated that after the GA crosslinking, collagen fibers become more organized and well-defined. Therefore, the preparations of porcine skin matrices with different degradation rates, which can be used in soft tissue reconstruction, are viable.

Proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus.

Science.gov (United States)

Balasubramanian, Sivaraman Arumugam; Mohan, Sujatha; Pye, David Cecil; Willcox, Mark Duncan Perry

2012-06-01

To investigate the expression of proteases, proteolytic activity and cytokines in the tear film of people with keratoconus. Basal tears from people with keratoconus, from individuals who had undergone corneal collagen cross-linking for the treatment of keratoconus, and from normal controls were collected using a capillary tube. Corneal curvature of each subject was mapped. The total protein in tears was estimated. Levels and activity of proteases in the tears were analysed using specific antibody arrays and activity assays. The total tear protein level was significantly reduced in keratoconus (4.1 ± 0.9 mg/ml) compared with normals (6.7 ± 1.4 mg/ml) (p tear expression of matrix metalloproteinases (MMP) -1, -3, -7, -13, interleukins (IL) -4, -5, -6, -8 and tumour necrosis factor (TNF) -α, -β were evident in keratoconus. Tear IL-6 was the only cytokine significantly (p tears of keratoconus subjects compared with the collagen cross-linked group. No significant difference in tear proteases were observed between the normal and the cross-linked groups, although the expression of TNF-α was significantly (p tears from keratoconus compared with normal subjects. The activity of tear gelatinases (69.6 ± 22.2 FIU) and collagenases (5.7 ± 3.3 FIU) in the collagen cross-linked group was not significantly different compared with either keratoconus or normals. Tears of people with keratoconus had 1.9 times higher levels of proteolytic activity and over expression of several MMPs and cytokines compared with tears from controls. Further investigations are required to study the possible implications of these changes and whether they can be used to monitor disease progression or determine the success of corneal collagen cross-linking. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Collagen derived serum markers in carcinoma of the prostate

DEFF Research Database (Denmark)

Rudnicki, M; Jensen, L T; Iversen, P

1995-01-01

Three new collagen markers deriving from the collagenous matrix, e.g. carboxyterminal propeptide of type I procollagen (PICP), carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP), and aminoterminal propeptide of type III procollagen (PIIINP) were used for the diagnose...

PERSONALIZED (LOCAL UV-CROSSLINKING AS A TREATMENT OF KERATOCONUS AND CORNEAL ECTASIA

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S. I. Anisimov

2017-01-01

Full Text Available There is no doubts that problem of keratoconus treatment is high relevant. The number of diagnosed ectasia, including iatrogenic ectasia, increased — in recent years. The amount of post-LASIK and ectasia after RK in late postoperative period multiplies significantly. It is associated not only with inception of new ways of treatment into practice, but also with the upgrade of diagnostic methods and equipment. A new method of local personalized crosslinking (LCXL was integrated into clinical practice using Laplace’s equation, pachymetry and topography maps data, It exceeds usual Dresden protocol on several parameters. This article shows the reaction of patients’ eyes on the treatment and the effectiveness of technique is provided. Materials and methods: topographic indications were evaluated (Orbscan II (B+L of 28 patients (40 eyes aged 18 — 44 with keratoconus stages I-IV (12 eyes — I stage; 16 eyes — II stage; 9 eyes — III stage; 3 eyes — IV stage classified by Abugova T.D., as well as UCVA, BCVA for 12 months after surgery. Normal values were estimated by characteristics of 30 eyes without any refractive errors. Results: LCXL is an effective way of treatment of corneal ectasia; it’s more effective in early stages. This technique has a strong controlled refractive impact and allows to reduce inflammatory after procedure. 

Corneal structure and transparency

Science.gov (United States)

Meek, Keith M.; Knupp, Carlo

2015-01-01

The corneal stroma plays several pivotal roles within the eye. Optically, it is the main refracting lens and thus has to combine almost perfect transmission of visible light with precise shape, in order to focus incoming light. Furthermore, mechanically it has to be extremely tough to protect the inner contents of the eye. These functions are governed by its structure at all hierarchical levels. The basic principles of corneal structure and transparency have been known for some time, but in recent years X-ray scattering and other methods have revealed that the details of this structure are far more complex than previously thought and that the intricacy of the arrangement of the collagenous lamellae provides the shape and the mechanical properties of the tissue. At the molecular level, modern technologies and theoretical modelling have started to explain exactly how the collagen fibrils are arranged within the stromal lamellae and how proteoglycans maintain this ultrastructure. In this review we describe the current state of knowledge about the three-dimensional stromal architecture at the microscopic level, and about the control mechanisms at the nanoscopic level that lead to optical transparency. PMID:26145225

Correlating rheological properties and printability of collagen bioinks: the effects of riboflavin photocrosslinking and pH.

Science.gov (United States)

Diamantides, Nicole; Wang, Louis; Pruiksma, Tylar; Siemiatkoski, Joseph; Dugopolski, Caroline; Shortkroff, Sonya; Kennedy, Stephen; Bonassar, Lawrence J

2017-07-05

Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8-9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.

Altered corneal stromal matrix organization is associated with mucopolysaccharidosis I, III and VI.

Science.gov (United States)

Alroy, J; Haskins, M; Birk, D E

1999-05-01

The presence of cloudy corneas is a prominent feature of mucopolysaccharidosis (MPS) types I and VI, but not MPS IIIA or IIIB. The cause of corneal cloudiness in MPS I and VI is speculative. Transparency of the cornea is dependent on the uniform diameter and the regular spacing and arrangement of the collagen fibrils within the stroma. Alterations in the spacing of collagen fibrils in a variety of conditions including corneal edema, scars, and macular corneal dystrophy is clinically manifested as corneal opacity. The purpose of this study was to compare the structural organization of the stromal extracellular matrix of normal corneas with that of MPS corneas. The size and arrangement of collagen fibrils in cloudy corneas from patients with MPS I were examined. The alterations observed were an increased mean fibril diameter with a broader distribution in the MPS corneas. The MPS I corneas also had altered fibril spacing and more irregular packing compared with normal control corneas. The clear corneas of patients with MPS IIIA and IIIB also showed increases in mean fibril diameter and fibril spacing. However, there was less variation indicating more regularity than seen in MPS I. In addition, corneas from cat models of certain MPS were compared to the human corneas. Cats with MPS I and VI, as well as normal control cats, were examined. Structural alterations comparable to those seen in human MPS corneas were seen in MPS I and VI cats relative to normal clear corneas. The findings suggest that cloudy corneas in MPS I and VI are in part a consequence of structural alterations in the corneal stroma, including abnormal spacing, size, and arrangement of collagen fibrils. Copyright 1999 Academic Press.

Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy.

Science.gov (United States)

Kim, Su-Young; Babar, Natasha; Munteanu, Emilia Laura; Takaoka, Anna; Zyablitskaya, Mariya; Nagasaki, Takayuki; Trokel, Stephen L; Paik, David C

2016-04-01

To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea. Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm. The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max. Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.

Visual rehabilitation in low-moderate keratoconus:intracorneal ring segment implantation followed by same-day topography-guided photorefractive keratectomy and collagen cross linking

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Ferial M. Zeraid

2014-10-01

Full Text Available AIM:To present the results of same-day topography-guided photorefractive keratectomy (TG-PRK and corneal collagen crosslinking (CXL after previous intrastromal corneal ring segment (ISCR implantation for keratoconus.METHODS:An experimental clinical study on twenty-one eyes of 19 patients aged 27.1±6.6y (range 19-43y, with low to moderate keratoconus who were selected to undergo customized TG-PRK immediately followed by same-day CXL, 9mo after ISCR implantation in a university ophthalmology clinic. Refraction, uncorrected distance visual acuities (UDVA and corrected distance visual acuities (CDVA, keratometry (K values, central corneal thickness (CCT and coma were assessed 3mo after TG-PRK and CXL.RESULTS:After TG-PRK/CXL:the mean UDVA (logMAR improved significantly from 0.66±0.41 to 0.20±0.25 (P<0.05; Kflat value decreased from:48.44±3.66 D to 43.71±1.95 D; Ksteep value decreased from 45.61±2.40 D to 41.56±2.05 D; Kaverage also decreased from 47.00±2.66 D to 42.42±2.07 D (P<0.05 for all. The mean sphere and cylinder decreased significantly post-surgery from, -3.10±2.99 D to -0.11±0.93 D and from -3.68±1.53 to -1.11±0.75 D respectively, while the CDVA, CCT and coma showed no significant changes. Compared to post-ISCR, significant reductions (P<0.05 or all in all K values, sphere and cylinder were observed after TG-PRK/CXL.CONCLUSION:Same-day combined topography-guided PRK and corneal crosslinking following placement of ISCR is a safe and potentially effective option in treating low-moderate keratoconus. It significantly improves all visual acuity, reduced keratometry, sphere and astigmatism, but causes no change in central corneal thickness and coma.

Applications of biomaterials in corneal wound healing

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I-Lun Tsai

2015-04-01

Full Text Available Disease affecting the cornea is a common cause of blindness worldwide. To date, the amniotic membrane (AM is the most widely used clinical method for cornea regeneration. However, donor-dependent differences in the AM may result in variable clinical outcomes. To overcome this issue, biomaterials are currently under investigation for corneal regeneration in vitro and in vivo. In this article, we highlight the recent advances in hydrogels, bioengineered prosthetic devices, contact lenses, and drug delivery systems for corneal regeneration. In clinical studies, the therapeutic effects of biomaterials, including fibrin and collagen-based hydrogels and silicone contact lenses, have been demonstrated in damaged cornea. The combination of cells and biomaterials may provide potential treatment in corneal wound healing in the future.

Clinical evaluation and induced corneal vascularization study by native and anionic collagen membranes in rabbits corneas Avaliação clínica e estudo da vascularização corneal induzida pelas membranas de colágeno nativo e aniônico em córneas de coelhos

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Thaís Eliane Binotto

2009-12-01

Full Text Available PURPOSE: To evaluate the corneal vascularization (CV and the clinical aspects induced by interlamellar graft with native (NCM and anionic (ACM collagen membranes in rabbits corneas. METHODS: An interlamellar graft with a 0.25 x 0.25 cm NCM (group 1 or ACM (group 2 fragment was performed in the right eye (treated eye. In the left eye, an estromal tunnel was done (control eye. Sixteen rabbits were used, and they were subdivided into two experimental groups of eight animals each. The clinical evaluation was performed at the 1st, 3rd, 7th, 15th and 30th postoperative days. Corneal vascularization analysis was performed after 30 days by the Images Analizator System Leica Qwin-550®. RESULTS: After 7 days, corneal vascularization was observed at about 2.25 ± 0.71 mm (NCM and at about 1.0 ± 1.69 mm (ACM, respectively, from the limbus in direction to the central cornea. After 15 days, CV increased in both groups (5.25 ± 1.03 mm - NCM; 2.0 ± 2.39 mm - ACM and then progressively decreased until day 30 (2.25 ± 2.10 mm - NCM; 0.75 ± 2.12 mm - ACM. The statistical analysis indicated that the averages of the distances from the limb vessels to the grafts observed after 7 and 15 days had not differed statistically (p=0.17, and after 15 and 30 postoperative days had a tendency to differ statistically (p=0.09. The control eyes did not present any changes. CONCLUSION: The interlamellar graft with native and anionic collagen membranes induced corneal vascularization when applied to rabbit corneas, but anionic collagen membrane induced a smaller corneal vascularization when compared to native collagen membrane. Although further studies are required, the results found in this study demonstrated the usefulness of interlamellar graft with native and anionic collagen membranes in keratoplasties. These membranes consists in one more graft option for the surgical treatment of corneal repair in rabbits and others animals, when other forms of medical and surgical

Comparing Change in Anterior Curvature after Corneal Cross-Linking Using Scanning-Slit and Scheimpflug Technology.

Science.gov (United States)

Lang, Paul Z; Thulasi, Praneetha; Khandelwal, Sumitra S; Hafezi, Farhad; Randleman, J Bradley

2018-05-02

To evaluate the correlation between anterior axial curvature difference maps following corneal cross-linking (CXL) for progressive keratoconus obtained from Scheimpflug-based tomography and Placido-based topography. Between-devices reliability analysis of randomized clinical trial data METHODS: Corneal imaging was collected at a single center institution pre-operatively and at 3, 6, and 12 months post-operatively using Scheimpflug-based tomography (Pentacam, Oculus Inc., Lynnwood, WA) and Scanning-slit, Placido-based topography (Orbscan II, Bausch & Lomb, Rochester, NY) in patients with progressive keratoconus receiving standard protocol CXL (3mW/cm 2 for 30 minutes). Regularization index (RI), absolute maximum keratometry (K Max), and change in (ΔK Max) were compared between the two devices at each time point. 51 eyes from 36 patients were evaluated at all time points. values were significantly different at all time points [56.01±5.3D Scheimpflug vs. 55.04±5.1D scanning-slit pre-operatively (p=0.003); 54.58±5.3D Scheimpflug vs. 53.12±4.9D scanning-slit at 12 months (pdevices (r=0.90-0.93) at all time points. The devices were not significantly different at any time point for either ΔK Max or RI but were poorly correlated at all time points (r=0.41-0.53 for ΔK Max, r=0.29-0.48 for RI). At 12 months, 95% LOA was 7.51D for absolute, 8.61D for ΔK Max, and 19.86D for RI. Measurements using Scheimpflug and scanning-slit Placido-based technology are correlated but not interchangeable. Both devices appear reasonable for separately monitoring the cornea's response to CXL; however, caution should be used when comparing results obtained with one measuring technology to the other. Copyright © 2018 Elsevier Inc. All rights reserved.

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Cabral, Wayne A; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Marini, Joan C; Yamauchi, Mitsuo

2017-08-04

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Corneal collagen cross-linking for progressive keratoconus in patients aged 9 to 14

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Fernando Plazzi Palis

2016-04-01

Full Text Available RESUMO Objetivo: Determinar a segurança e eficácia do cross-linking corneano (CXL em pacientes de 9 a 14 anos de idade com ceratocone progressivo. Métodos: Dezesseis olhos de onze pacientes (8 homens e 3 mulheres com ceratocone progressivo foram submetidos ao CXL, de acordo com o protocolo padrão de Dresden. A média do tempo de seguimento foi de 26 meses (variando de 12 a 60 meses. Os exames pré e pós-operatórios incluíram: acuidade visual sem correção (AVSC, melhor acuidade visual com correção (AVCC, topografia corneana, tonometria, refração, paquimetria corneana, e contagem de células endoteliais. Resultados: Na última visita de acompanhamento ambulatorial, a AVCC melhorou pelo menos uma linha na tabela de Snellen em 6 olhos (37,5% e permaneceu estável em 9 olhos (56,25%. Dois olhos (12,5% de pacientes que coçam os olhos com frequência, exigiram retratamento devido à progressão do ceratocone, 15 e 28 meses após o primeiro CXL. A refração e contagem de células endoteliais mantiveram-se estáveis. Os resultados topográficos mostraram melhora estatisticamente significativa nos valores do K máximo até dois anos após o CXL. No entanto, houve perda de significância ao longo do tempo. Nenhuma complicação peroperatória foi observada. Dois olhos (12,5% apresentaram haze grau I, que regrediu após um mês de terapia com esteróide tópico. Conclusão: Neste estudo com pacientes selecionados, de 9 a 14 anos de idade, o CXL mostrou ser uma opção segura e eficaz para o tratamento do ceratocone progressivo. No entanto, o efeito pode não ser duradouro, podendo ser necessário um novo tratamento. Maior amostragem e maior seguimento são necessários para verificar esta tendência.

Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

Energy Technology Data Exchange (ETDEWEB)

Zhong Shaoping [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Teo, Wee Eong [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Zhu Xiao [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Beuerman, Roger [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Ramakrishna, Seeram [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Yung, Lin Yue Lanry [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore)]. E-mail: cheyly@nus.edu.sg

2007-03-15

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.

Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

International Nuclear Information System (INIS)

Zhong Shaoping; Teo, Wee Eong; Zhu Xiao; Beuerman, Roger; Ramakrishna, Seeram; Yung, Lin Yue Lanry

2007-01-01

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds

The effects of different cross-linking conditions on collagen-based nanocomposite scaffolds—an in vitro evaluation using mesenchymal stem cells

International Nuclear Information System (INIS)

Suchý, Tomáš; Šupová, Monika; Sucharda, Zbyněk; Rýglová, Šárka; Žaloudková, Margit; Sauerová, Pavla; Kalbáčová, Marie Hubálek; Verdánová, Martina; Sedláček, Radek

2015-01-01

Nanocomposite scaffolds which aimed to imitate a bone extracellular matrix were prepared for bone surgery applications. The scaffolds consisted of polylactide electrospun nano/sub-micron fibres, a natural collagen matrix supplemented with sodium hyaluronate and natural calcium phosphate nano-particles (bioapatite). The mechanical properties of the scaffolds were improved by means of three different cross-linking agents: N-(3-dimethylamino propyl)-N’-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an ethanol solution (EDC/NHS/EtOH), EDC/NHS in a phosphate buffer saline solution (EDC/NHS/PBS) and genipin. The effect of the various cross-linking conditions on the pore size, structure and mechanical properties of the scaffolds were subsequently studied. In addition, the mass loss, the swelling ratio and the pH of the scaffolds were determined following their immersion in a cell culture medium. Furthermore, the metabolic activity of human mesenchymal stem cells (hMSCs) cultivated in scaffold infusions for 2 and 7 days was assessed. Finally, studies were conducted of cell adhesion, proliferation and penetration into the scaffolds. With regard to the structural stability of the tested scaffolds, it was determined that EDC/NHS/PBS and genipin formed the most effectively cross-linked materials. Moreover, it was discovered that the genipin cross-linked scaffold also provided the best conditions for hMSC cultivation. In addition, the infusions from all the scaffolds were found to be non-cytotoxic. Thus, the genipin and EDC/NHS/PBS cross-linked scaffolds can be considered to be promising biomaterials for further in vivo testing and bone surgery applications. (paper)

A histological study of rabbit corneas after transepithelial corneal crosslinking using partial epithelial photoablation or ethanol treatment.

Science.gov (United States)

Ozmen, Mehmet Cuneyt; Hondur, Ahmet; Yilmaz, Guldal; Bilgihan, Kamil; Hasanreisoglu, Berati

2014-01-01

To evaluate the histological changes after transepithelial corneal crosslinking (CXL) using partial thickness excimer laser ablation or epithelial ethanol application in an experimental rabbit study. Right eyes of twenty-four rabbits were studied. Four eyes received total epithelial debridement (group I). Four eyes received partial thickness epithelial ablation with excimer laser (group II). Twelve eyes were treated with different durations (30s and 60s) and concentrations (18% to 48%) of ethanol (group III). Riboflavin was applied for 30min intervals along with topical proparacaine drops with benzalkonium chloride, and 370 nm irradiation was performed for 30min, while riboflavin was instilled every 3min. Four eyes (group IV) received 48% ethanol for 30s without riboflavin and irradiation. Eyes were collected after 24h and examined histologically. All eyes in group I showed keratocyte loss in the superficial 300 µ of corneal storma. In group II, 1-4 layers of epithelium were preserved and no keratocyte loss occurred. In group III, CXL after treatment with ethanol up to 24% concentration and up to 60s revealed no keratocyte loss. CXL after treatment with 48% and higher ethanol concentrations yielded keratocyte loss in the superficial 200 µ to 300 µ of cornea. Incomplete excimer laser ablation of the epithelium or treatment with ethanol up to 24% concentration and up to 60s duration yielded no stromal keratocyte loss. To get the same histological appearance seen in epithelial debridement group, partial thickness excimer laser epithelial ablation or ethanol application is not adequate for transepithelial CXL.

A histological study of rabbit corneas after transepithelial corneal crosslinking using partial epithelial photoablation or ethanol treatment

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Mehmet Cuneyt Ozmen

2014-12-01

Full Text Available AIM: To evaluate the histological changes after transepithelial corneal crosslinking (CXL using partial thickness excimer laser ablation or epithelial ethanol application in an experimental rabbit study.METHODS: Right eyes of twenty-four rabbits were studied. Four eyes received total epithelial debridement (group I. Four eyes received partial thickness epithelial ablation with excimer laser (group II. Twelve eyes were treated with different durations (30s and 60s and concentrations (18% to 48% of ethanol (group III. Riboflavin was applied for 30min intervals along with topical proparacaine drops with benzalkonium chloride, and 370 nm irradiation was performed for 30min, while riboflavin was instilled every 3min. Four eyes (group IV received 48% ethanol for 30s without riboflavin and irradiation. Eyes were collected after 24h and examined histologically.RESULTS: All eyes in group I showed keratocyte loss in the superficial 300 µ of corneal storma. In group II, 1-4 layers of epithelium were preserved and no keratocyte loss occurred. In group III, CXL after treatment with ethanol up to 24% concentration and up to 60s revealed no keratocyte loss. CXL after treatment with 48% and higher ethanol concentrations yielded keratocyte loss in the superficial 200 µ to 300 µ of cornea.CONCLUSION: Incomplete excimer laser ablation of the epithelium or treatment with ethanol up to 24% concentration and up to 60s duration yielded no stromal keratocyte loss. To get the same histological appearance seen in epithelial debridement group, partial thickness excimer laser epithelial ablation or ethanol application is not adequate for transepithelial CXL.

Cross-linking of collagen-based materials

NARCIS (Netherlands)

Zeeman, R.

1998-01-01

An example of a collagen-based tissue is the aortic heart valve. A variety of pathological processes can lead to heart valve malfunction and this is usually associated with degenerative changes of the tissue. The most commonly used types of prosthetic valves are mechanical and tissue valves. One

The roles of TGF-beta1 gene transfer on collagen formation during Achilles tendon healing.

Science.gov (United States)

Hou, Yu; Mao, ZeBing; Wei, XueLei; Lin, Lin; Chen, LianXu; Wang, HaiJun; Fu, Xin; Zhang, JiYing; Yu, ChangLong

2009-05-29

Collagen content and cross-linking are believed to be major determinants of tendon structural integrity and function. The current study aimed to investigate the effects of transforming growth factor (TGF)-beta1 on the collagen content and cross-linking of Achilles tendons, and on the histological and biomechanical changes occurring during Achilles tendon healing in rabbits. Bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the TGF-beta1 gene were surgically implanted into experimentally injured Achilles tendons. Collagen proteins were identified by immunohistochemical staining and fiber bundle accumulation was revealed by Sirius red staining. Achilles tendons treated with TGF-beta1-transfected BMSCs showed higher concentrations of collagen I protein, more rapid matrix remodeling, and larger fiber bundles. Thus TGF-beta1 can promote mechanical strength in healing Achilles tendons by regulating collagen synthesis, cross-link formation, and matrix remodeling.

Enhanced stabilization of collagen by furfural.

Science.gov (United States)

Lakra, Rachita; Kiran, Manikantan Syamala; Usha, Ramamoorthy; Mohan, Ranganathan; Sundaresan, Raja; Korrapati, Purna Sai

2014-04-01

Furfural (2-furancarboxaldehyde), a product derived from plant pentosans, has been investigated for its interaction with collagen. Introduction of furfural during fibril formation enhanced the thermal and mechanical stability of collagen. Collagen films treated with furfural exhibited higher denaturation temperature (Td) (pFurfural and furfural treated collagen films did not have any cytotoxic effect. Rheological characterization showed an increase in shear stress and shear viscosity with increasing shear rate for treated collagen. Circular dichroism (CD) studies indicated that the furfural did not have any impact on triple helical structure of collagen. Scanning electron microscopy (SEM) of furfural treated collagen exhibited small sized porous structure in comparison with untreated collagen. Thus this study provides an alternate ecologically safe crosslinking agent for improving the stability of collagen for biomedical and industrial applications. Copyright © 2014 Elsevier B.V. All rights reserved.

A three-dimensional computational model of collagen network mechanics.

Directory of Open Access Journals (Sweden)

Byoungkoo Lee

Full Text Available Extracellular matrix (ECM strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned. We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.

A Rare Form of Corneal Opacity Associated with Spondyloepiphyseal Dysplasia Congenita

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Yuichiro Ishida

2018-02-01

Full Text Available A 13-year-old Japanese female diagnosed with spondyloepiphyseal dysplasia congenita (SEDC was referred for ophthalmologic evaluation. Examination with slit-lamp and optical coherence tomography revealed bilateral thin cornea with diffuse corneal opacity which was localised at the posterior stromal depth in the central cornea. Unlike the two previously reported cases of diffuse and nodular patterns of corneal opacity in SEDC, the current case exhibited a rare form of corneal opacity. SEDC is one of the type II collagenopathies, characterised by dwarfism because the mutations in COL2A1 prevent bone growth. Although the existence of type II collagen has not been reported in the human corneal stroma, the aetiology of the opacity in the corneal stroma in SEDC type II collagenopathy is of interest.

The chemical reactivity and structure of collagen studied by neutron diffraction

Energy Technology Data Exchange (ETDEWEB)

Wess, T.J.; Wess, L.; Miller, A. [Univ. of Stirling (United Kingdom)

1994-12-31

The chemical reactivity of collagen can be studied using neutron diffraction (a non-destructive technique), for certain reaction types. Collagen contains a number of lysine and hydroxylysine side chains that can react with aldehydes and ketones, or these side chains can themselves be converted to aldehydes by lysyl oxidase. The reactivity of these groups not only has an important role in the maintenance of mechanical strength in collagen fibrils, but can also manifest pathologically in the cases of aging, diabetes (reactivity with a variety of sugars) and alcoholism (reactivity with acetaldehyde). The reactivity of reducing groups with collagen can be studied by neutron diffraction, since the crosslink formed in the adduction process is initially of a Schiff base or keto-imine nature. The nature of this crosslink allows it to be deuterated, and the position of this relatively heavy scattering atom can be used in a process of phase determination by multiple isomorphous replacement. This process was used to study the following: the position of natural crosslinks in collagen; the position of adducts in tendon from diabetic rats in vivo and the in vitro position of acetaidehyde adducts in tendon.

The chemical reactivity and structure of collagen studied by neutron diffraction

International Nuclear Information System (INIS)

Wess, T.J.; Wess, L.; Miller, A.

1994-01-01

The chemical reactivity of collagen can be studied using neutron diffraction (a non-destructive technique), for certain reaction types. Collagen contains a number of lysine and hydroxylysine side chains that can react with aldehydes and ketones, or these side chains can themselves be converted to aldehydes by lysyl oxidase. The reactivity of these groups not only has an important role in the maintenance of mechanical strength in collagen fibrils, but can also manifest pathologically in the cases of aging, diabetes (reactivity with a variety of sugars) and alcoholism (reactivity with acetaldehyde). The reactivity of reducing groups with collagen can be studied by neutron diffraction, since the crosslink formed in the adduction process is initially of a Schiff base or keto-imine nature. The nature of this crosslink allows it to be deuterated, and the position of this relatively heavy scattering atom can be used in a process of phase determination by multiple isomorphous replacement. This process was used to study the following: the position of natural crosslinks in collagen; the position of adducts in tendon from diabetic rats in vivo and the in vitro position of acetaidehyde adducts in tendon

The Use of Polymerized Genipin for the Stabilization of the Collagen Structure of Animal Hides

Science.gov (United States)

Animal hides are the major byproduct of meat industry and the collagen fibers is the main constituent. Crosslinkers play a key role in stabilizing the collagen structure for useful applications. Genipin is widely used as an ideal biological protein crosslinking agent due to its low toxicity compare...

Can green solvents be alternatives for thermal stabilization of collagen?

Science.gov (United States)

Mehta, Ami; Rao, J Raghava; Fathima, Nishter Nishad

2014-08-01

"Go Green" campaign is gaining light for various industrial applications where water consumption needs to be reduced. To resolve this, industries have adopted usage of green, organic solvents, as an alternative to water. For leather making, tanning industry consumes gallons of water. Therefore, for adopting green solvents in leather making, it is necessary to evaluate its influence on type I collagen, the major protein present in the skin matrix. The thermal stability of collagen from rat tail tendon fiber (RTT) treated with seven green solvents namely, ethanol, ethyl lactate, ethyl acetate, propylene carbonate, propylene glycol, polyethylene glycol-200 and heptane was determined using differential scanning calorimetry (DSC). Crosslinking efficiency of basic chromium sulfate and wattle on RTT in green solvents was determined. DSC thermograms show increase in thermal stability of RTT collagen against heat with green solvents (>78°C) compared to water (63°C). In the presence of crosslinkers, RTT demonstrated thermal stability >100°C in some green solvents, resulting in increased intermolecular forces between collagen, solvent and crosslinkers. The significant improvement in thermal stability of collagen potentiates the capability of green solvents as an alternative for water. Copyright © 2014 Elsevier B.V. All rights reserved.

Collagen turnover after tibial fractures

DEFF Research Database (Denmark)

Joerring, S; Krogsgaard, M; Wilbek, H

1994-01-01

Collagen turnover after tibial fractures was examined in 16 patients with fracture of the tibial diaphysis and in 8 patients with fracture in the tibial condyle area by measuring sequential changes in serological markers of turnover of types I and III collagen for up to 26 weeks after fracture....... The markers were the carboxy-terminal extension peptide of type I procollagen (PICP), the amino-terminal extension peptide of type III procollagen (PIIINP), and the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP). The latter is a new serum marker of degradation of type I...... collagen. A group comparison showed characteristic sequential changes in the turnover of types I and III collagen in fractures of the tibial diaphysis and tibial condyles. The turnover of type III collagen reached a maximum after 2 weeks in both groups. The synthesis of type I collagen reached a maximum...

Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

Science.gov (United States)

Eyre, David R.; Ann Weis, Mary

2013-01-01

Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.

Science.gov (United States)

Xu, Bin; Song, Zhan; Fan, Tingjun

2017-11-01

Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

MICROWAVE IRRADIATION AND CROSS-LINKING OF COLLAGEN

NARCIS (Netherlands)

VISSER, CE; VOUTE, ABE; OOSTING, J; BOON, ME; KOK, LP

1992-01-01

In a multifactorial experiment, dermal sheep collagen was treated in diluted glutaraldehyde solutions, 70% ethyl alcohol, Cialit 1:5000, and distilled water for 1, 3 and 5 min, respectively, in combination with microwave irradiation at different temperature settings. The shrinkage temperature

Unilateral congenital corneal keloid with anterior segment mesenchymal dysgenesis and subluxated lens: case report and review of literature.

Science.gov (United States)

Vanathi, M; Sen, Seema; Panda, Anita; Dada, Tanuj; Behera, Geeta; Khokhar, Sudharshan

2007-01-01

To report the unusual association of unilateral congenital corneal keloid with anterior-segment mesenchymal dysgenesis and bilateral subluxated lens. A 20-year old man presented with a mass lesion involving the left cornea. The corneal lesion had been present since birth. On biomicroscopic examination, a well-defined vascularized, grayish-white mass occupying the whole of the left cornea was seen. The right eye showed multiple peripheral corneal opacities with iridocorneal adhesions, a poorly defined supranasal limbus, and a subluxated lens. Excision biopsy of the mass was done for histopathologic examination. Histopathologic examination of the excised corneal mass showed features consistent with that of a corneal keloid: thickened keratinized epithelium, absent Bowman membrane layer, and fibrovascular hyperplasia composed of hyalinized collagen fibers with irregular orientation of the collagen lamellae. During penetrating keratoplasty of the left eye, an anomalous iris pattern with poorly defined angle and a supranasal subluxated lens was also observed. Extraction of the subluxated lens was also done. The graft failed subsequent to a nonhealing persistent epithelial defect. Our case report highlights the rare association of a unilateral congenital corneal keloid with anterior-segment mesenchymal dysgenesis and bilateral subluxated lens.

Physiochemical properties and resorption progress of porcine skin-derived collagen membranes: In vitro and in vivo analysis.

Science.gov (United States)

An, Yin-Zhe; Kim, You-Kyoung; Lim, Su-Min; Heo, Yeong-Ku; Kwon, Mi-Kyung; Cha, Jae-Kook; Lee, Jung-Seok; Jung, Ui-Won; Choi, Seong-Ho

2018-03-30

The aim of the present study was to evaluate the physiochemical properties and resorption progress of two cross-linked, porcine skin-derived collagen membranes and compare their features with those of a membrane without cross-linking (Bio-Gide ® [BG], Geistlich Biomaterials, Wolhusen, Switzerland). Three porcine skin-derived collagen membranes, dehydrothermally (DHT) cross-linked (experimental), DHT and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (DHT/EDC) cross-linked (experimental) and BG were investigated for their morphology, enzyme resistance, and tensile strength in vitro and biodegradation in vivo. DHT and DHT/EDC membranes exhibited irregular, interconnected macro- and micropores that formed a 3D mesh, whereas BG exhibited individual collagen fibrils interlaced to form coarse collagen strands. In enzyme resistance and tensile strength tests, DHT and DHT/EDC membranes demonstrated good resistance and mechanical properties compared with BG. In vivo, all three membranes were well integrated into the surrounding connective tissue. Thus, the DHT membrane exhibited its potential as a barrier membrane for guided bone and tissue regeneration.

Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance

Science.gov (United States)

Hapach, Lauren A.; VanderBurgh, Jacob A.; Miller, Joseph P.; Reinhart-King, Cynthia A.

2015-12-01

Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

Glutaraldehyde-induced remineralization improves the mechanical properties and biostability of dentin collagen

Energy Technology Data Exchange (ETDEWEB)

Chen, Chaoqun; Mao, Caiyun; Sun, Jian; Chen, Yi; Wang, Wei [Department of Stomatology, The First Affiliated Hospital, College of Medicine, Zhejiang University (China); Pan, Haihua; Tang, Ruikang [Centre for Biopathways and Biomaterials, Department of Chemistry, Zhejiang University (China); Gu, Xinhua [Department of Stomatology, The First Affiliated Hospital, College of Medicine, Zhejiang University (China)

2016-10-01

The purpose of this study was to induce a biomimetic remineralization process by using glutaraldehyde (GA) to reconstruct the mechanical properties and biostability of demineralized collagen. Demineralized dentin disks (35% phosphoric acid, 10 s) were pretreated with a 5% GA solution for 3 min and then cultivated in a calcium phosphate remineralization solution. The remineralization kinetics and superstructure of the remineralization layer were evaluated by Raman spectroscopy, transmission electron microscopy, scanning electron microscopy and nanoindentation tests. The biostability was examined by enzymatic degradation experiments. A significant difference was found in dentin remineralization process between dentin with and without GA pretreating. GA showed a specific affinity to dentin collagen resulting in the formation of a cross-linking superstructure. GA pretreating could remarkably shorten remineralization time from 7 days to 2 days. The GA-induced remineralized collagen fibrils were well encapsulated by newly formed hydroxyapatite mineral nanocrystals. With the nano-hydroxyapatite coating, both the mechanical properties (elastic modulus and hardness) and the biostability against enzymatic degradation of the collagen were significantly enhanced, matching those of natural dentin. The results indicated that GA cross-linking of dentin collagen could promote dentin biomimetic remineralization, resulting in an improved mechanical properties and biostability. It may provide a promising tissue-engineering technology for dentin repair. - Highlights: • GA cross-linking can promote the remineralization kinetics of dentin collagen. • GA-induced remineralization can reshape the demineralized dentin collagen layer. • The GA-induced remineralization enhances the degradation resistance of collagen. • GA-induced remineralization provides a new approach to improve bonding durability.

Glutaraldehyde-induced remineralization improves the mechanical properties and biostability of dentin collagen

International Nuclear Information System (INIS)

Chen, Chaoqun; Mao, Caiyun; Sun, Jian; Chen, Yi; Wang, Wei; Pan, Haihua; Tang, Ruikang; Gu, Xinhua

2016-01-01

The purpose of this study was to induce a biomimetic remineralization process by using glutaraldehyde (GA) to reconstruct the mechanical properties and biostability of demineralized collagen. Demineralized dentin disks (35% phosphoric acid, 10 s) were pretreated with a 5% GA solution for 3 min and then cultivated in a calcium phosphate remineralization solution. The remineralization kinetics and superstructure of the remineralization layer were evaluated by Raman spectroscopy, transmission electron microscopy, scanning electron microscopy and nanoindentation tests. The biostability was examined by enzymatic degradation experiments. A significant difference was found in dentin remineralization process between dentin with and without GA pretreating. GA showed a specific affinity to dentin collagen resulting in the formation of a cross-linking superstructure. GA pretreating could remarkably shorten remineralization time from 7 days to 2 days. The GA-induced remineralized collagen fibrils were well encapsulated by newly formed hydroxyapatite mineral nanocrystals. With the nano-hydroxyapatite coating, both the mechanical properties (elastic modulus and hardness) and the biostability against enzymatic degradation of the collagen were significantly enhanced, matching those of natural dentin. The results indicated that GA cross-linking of dentin collagen could promote dentin biomimetic remineralization, resulting in an improved mechanical properties and biostability. It may provide a promising tissue-engineering technology for dentin repair. - Highlights: • GA cross-linking can promote the remineralization kinetics of dentin collagen. • GA-induced remineralization can reshape the demineralized dentin collagen layer. • The GA-induced remineralization enhances the degradation resistance of collagen. • GA-induced remineralization provides a new approach to improve bonding durability.

Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

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Marwa Kamel

2017-01-01

Full Text Available The role of the extracellular matrix (ECM in uterine fibroids (UF has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP and lysylpyridinoline (LP cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH and lysyl oxidases (LOX], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b and LOX. Also, increased resistance to matrix metalloproteinases (MMP proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA, growth-promoting markers (PCNA; pERK1/2; FAKpY397; Ki-67; and Cyclin D1, and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

The ratio 1660/1690 cm(-1) measured by infrared microspectroscopy is not specific of enzymatic collagen cross-links in bone tissue.

Science.gov (United States)

Farlay, Delphine; Duclos, Marie-Eve; Gineyts, Evelyne; Bertholon, Cindy; Viguet-Carrin, Stéphanie; Nallala, Jayakrupakar; Sockalingum, Ganesh D; Bertrand, Dominique; Roger, Thierry; Hartmann, Daniel J; Chapurlat, Roland; Boivin, Georges

2011-01-01

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm(-1) area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm(-1) by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm(-1) by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm(-1) area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm(-1) ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm(-1) was unmodified. In conclusion, the 1660/1690 cm(-1) is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process.

The Ratio 1660/1690 cm−1 Measured by Infrared Microspectroscopy Is Not Specific of Enzymatic Collagen Cross-Links in Bone Tissue

Science.gov (United States)

Farlay, Delphine; Duclos, Marie-Eve; Gineyts, Evelyne; Bertholon, Cindy; Viguet-Carrin, Stéphanie; Nallala, Jayakrupakar; Sockalingum, Ganesh D.; Bertrand, Dominique; Roger, Thierry; Hartmann, Daniel J.; Chapurlat, Roland; Boivin, Georges

2011-01-01

In postmenopausal osteoporosis, an impairment in enzymatic cross-links (ECL) occurs, leading in part to a decline in bone biomechanical properties. Biochemical methods by high performance liquid chromatography (HPLC) are currently used to measure ECL. Another method has been proposed, by Fourier Transform InfraRed Imaging (FTIRI), to measure a mature PYD/immature DHLNL cross-links ratio, using the 1660/1690 cm−1 area ratio in the amide I band. However, in bone, the amide I band composition is complex (collagens, non-collagenous proteins, water vibrations) and the 1660/1690 cm−1 by FTIRI has never been directly correlated with the PYD/DHLNL by HPLC. A study design using lathyritic rats, characterized by a decrease in the formation of ECL due to the inhibition of lysyl oxidase, was used in order to determine the evolution of 1660/1690 cm−1 by FTIR Microspectroscopy in bone tissue and compare to the ECL quantified by HPLC. The actual amount of ECL was quantified by HPLC on cortical bone from control and lathyritic rats. The lathyritic group exhibited a decrease of 78% of pyridinoline content compared to the control group. The 1660/1690 cm−1 area ratio was increased within center bone compared to inner bone, and this was also correlated with an increase in both mineral maturity and mineralization index. However, no difference in the 1660/1690 cm−1 ratio was found between control and lathyritic rats. Those results were confirmed by principal component analysis performed on multispectral infrared images. In bovine bone, in which PYD was physically destructed by UV-photolysis, the PYD/DHLNL (measured by HPLC) was strongly decreased, whereas the 1660/1690 cm−1 was unmodified. In conclusion, the 1660/1690 cm−1 is not related to the PYD/DHLNL ratio, but increased with age of bone mineral, suggesting that a modification of this ratio could be mainly due to a modification of the collagen secondary structure related to the mineralization process. PMID:22194900

The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells.

Science.gov (United States)

Zhou, Chengxin; Lei, Fengyang; Chodosh, James; Paschalis, Eleftherios I

2016-04-01

Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.

The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus aureus Keratitis

Science.gov (United States)

Rhem, Marcus N.; Lech, Elizabeth M.; Patti, Joseph M.; McDevitt, Damien; Höök, Magnus; Jones, Dan B.; Wilhelmus, Kirk R.

2000-01-01

A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea. PMID:10816547

5-year follow-up of combined non-topography guided photorefractive keratectomy and corneal collagen cross linking for keratoconus

Directory of Open Access Journals (Sweden)

Abdulrahman Mohammed Al-Amri

2018-01-01

Full Text Available AIM: To evaluate the visual outcomes of simultaneous non-topography guided photorefractive keratectomy (PRK and corneal collagen cross-linking (CXL in eyes with keratoconus 5y after the procedure. METHODS: Prospective, interventional, non-randomized, and non-controlled case series design was used. Sixty eyes of 30 patients (16 males and 14 females; age: 21-41y with mild, non-progressive (stages 1-2 keratoconus were enrolled. Refraction, uncorrected distance visual acuity (UDVA and corrected distance visual acuity (CDVA , flat and steep keratometry readings, and adverse events were evaluated preoperatively and postoperatively. Data were collected preoperatively and postoperatively at 3mo, 1, 2, 3, 4, and 5y follow-up visits after combined non-topography-guided PRK with CXL was performed. All patients had at least 5y of follow-up. RESULTS: All study parameters showed a statistically significant improvement at 5y over baseline values. The mean follow-up time was 68.20±4.71mo (range: 60-106mo. Patients showed a significant improvement in UDVA from 1.24±0.79 logMAR prior to combined non-TG-PRK+CXL to 0.06±0.15 logMAR postoperatively at the time of their last follow-up visit. CDVA significantly increased from 0.06±0.19 logMAR preoperatively to 0.03±0.12 logMAR postoperatively. A significant decrease in the mean spherical equivalent (SE refraction was observed from -2.28±1.8 to -0.79±0.93 diopters (D (P<0.05, and the manifest sphere decreased from -1.62±1.23 to -0.27±0.21 D (P=0.001. The manifest cylinder significantly decreased from -1.73±0.86 to -0.29±0.34 D postoperatively (P=0.001. The mean steep keratometry was 45.13±1.32 vs 47.28±2.12 D preoperatively (P<0.05, and the preoperative mean steepest keratometry (Kmax 48.6±3.1 was reduced significantly to 46.8±2.9 postoperatively (P<0.05. CONCLUSION: Combined non-TG-PRK with 15min CXL is an effective and safe option for correcting mild refractive error and improving visual acuity in

Collagen derived serum markers in carcinoma of the prostate

DEFF Research Database (Denmark)

Rudnicki, M; Jensen, L T; Iversen, P

1995-01-01

Three new collagen markers deriving from the collagenous matrix, e.g. carboxyterminal propeptide of type I procollagen (PICP), carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP), and aminoterminal propeptide of type III procollagen (PIIINP) were used for the diagnose......, ICTP, and PICP did not differ between these two groups. In patients with metastatic prostatic cancer all five markers were increased compared to the level measured in patients with localized cancer (p

The Antifibrosis Effects of Peroxisome Proliferator-Activated Receptor δ on Rat Corneal Wound Healing after Excimer Laser Keratectomy

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Yun Gu

2014-01-01

Full Text Available Corneal stromal fibrosis characterized by myofibroblasts and abnormal extracellular matrix (ECM is usually the result of inappropriate wound healing. The present study tested the hypothesis that the ligand activation of peroxisome proliferator-activated receptor (PPAR δ had antifibrosis effects in a rat model of corneal damage. Adult Sprague-Dawley rats underwent bilateral phototherapeutic keratectomy (PTK. The eyes were randomized into four groups: PBS, GW501516 (a selective agonist of PPARδ, GSK3787 (a selective antagonist of PPARδ, or GW501516 combined with GSK3787. The agents were subconjunctivally administered twice a week until sacrifice. The cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and postmortem histology. A myofibroblast marker (α-smooth muscle actin and ECM production (fibronectin, collagen type III and collagen type I were examined by immunohistochemistry and RT-PCR. At the early stages of wound healing, GW501516 inhibited reepithelialization and promoted angiogenesis. During the remodeling phase of wound healing, GW501516 attenuated the activation and proliferation of keratocytes, which could be reversed by GSK3787. GW501516 decreased transdifferentiation from keratocytes into myofibroblasts, ECM synthesis, and corneal haze. These results demonstrate that GW501516 controls corneal fibrosis and suggest that PPARδ may potentially serve as a therapeutic target for treating corneal scars.

Biocompatibility and Biomechanical Effect of Single Wall Carbon Nanotubes Implanted in the Corneal Stroma: A Proof of Concept Investigation

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Alfredo Vega-Estrada

2016-01-01

Full Text Available Corneal ectatic disorders are characterized by a progressive weakening of the tissue due to biomechanical alterations of the corneal collagen fibers. Carbon nanostructures, mainly carbon nanotubes (CNTs and graphene, are nanomaterials that offer extraordinary mechanical properties and are used to increase the rigidity of different materials and biomolecules such as collagen fibers. We conducted an experimental investigation where New Zealand rabbits were treated with a composition of CNTs suspended in balanced saline solution which was applied in the corneal tissue. Biocompatibility of the composition was assessed by means of histopathology analysis and mechanical properties by stress-strain measurements. Histopathology samples stained with blue Alcian showed that there were no fibrous scaring and no alterations in the mucopolysaccharides of the stroma. It also showed that there were no signs of active inflammation. These were confirmed when Masson trichrome staining was performed. Biomechanical evaluation assessed by means of tensile test showed that there is a trend to obtain higher levels of rigidity in those corneas implanted with CNTs, although these changes are not statistically significant (p>0.05. Implanting CNTs is biocompatible and safe procedure for the corneal stroma which can lead to an increase in the rigidity of the collagen fibers.

Fabrication and evaluation of biomimetic scaffolds by using collagen-alginate fibrillar gels for potential tissue engineering applications

International Nuclear Information System (INIS)

Sang Lin; Luo Dongmei; Xu Songmei; Wang Xiaoliang; Li Xudong

2011-01-01

Pore architecture and its stable functionality under cell culturing of three dimensional (3D) scaffolds are of great importance for tissue engineering purposes. In this study, alginate was incorporated with collagen to fabricate collagen-alginate composite scaffolds with different collagen/alginate ratios by lyophilizing the respective composite gels formed via collagen fibrillogenesis in vitro and then chemically crosslinking. The effects of alginate amount and crosslinking treatment on pore architecture, swelling behavior, enzymatic degradation and tensile property of composite scaffolds were systematically investigated. The relevant results indicated that the present strategy was simple but efficient to fabricate highly interconnected strong biomimetic 3D scaffolds with nanofibrous surface. NIH3T3 cells were used as a model cell to evaluate the cytocompatibility, attachment to the nanofibrous surface and porous architectural stability in terms of cell proliferation and infiltration within the crosslinked scaffolds. Compared with the mechanically weakest crosslinked collagen sponges, the cell-cultured composite scaffolds presented a good porous architecture, thus permitting cell proliferation on the top surface as well as infiltration into the inner part of 3D composite scaffolds. These composite scaffolds with pore size ranging from 150 to 300 μm, over 90% porosity, tuned biodegradability and water-uptake capability are promising for tissue engineering applications.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.

Science.gov (United States)

Sorkio, Anni; Koch, Lothar; Koivusalo, Laura; Deiwick, Andrea; Miettinen, Susanna; Chichkov, Boris; Skottman, Heli

2018-07-01

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted

From Nano to Macro: Studying the Hierarchical Structure of the Corneal Extracellular Matrix

Science.gov (United States)

Quantock, Andrew J.; Winkler, Moritz; Parfitt, Geraint J.; Young, Robert D.; Brown, Donald J.; Boote, Craig; Jester, James V.

2014-01-01

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the ‘nano’ to the ‘macro’ levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometer level using x-ray scattering through to the millimeter to centimeter level using nonlinear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering. PMID:25819457

Shear bond strength of composite to deep dentin after treatment with two different collagen cross-linking agents at varying time intervals.

Science.gov (United States)

Srinivasulu, S; Vidhya, S; Sujatha, M; Mahalaxmi, S

2012-01-01

This in vitro study evaluated the shear bond strength of composite resin to deep dentin using a total etch adhesive after treatment with two collagen cross-linking agents at varying time intervals. Thirty freshly extracted human maxillary central incisors were sectioned longitudinally into equal mesial and distal halves (n=60). The proximal deep dentin was exposed, maintaining a remaining dentin thickness (RDT) of approximately 1 mm. The specimens were randomly divided into three groups based on the surface treatment of dentin prior to bonding as follows: group I (n=12, control): no prior dentin surface treatment; group II (n=24): dentin surface pretreated with 10% sodium ascorbate; and group III (n=24): dentin surface pretreated with 6.5% proanthocyanidin. Groups II and III were further subdivided into two subgroups of 12 specimens each, based on the pretreatment time of five minutes (subgroup A) and 10 minutes (subgroup B). Shear bond strength of the specimens was tested with a universal testing machine, and the data were statistically analyzed. Significantly higher shear bond strength to deep dentin was observed in teeth treated with 10% sodium ascorbate (group II) and 6.5% proanthocyanidin (group III) compared to the control group (group I). Among the collagen cross-linkers used, specimens treated with proanthocyanidin showed significantly higher shear bond strength values than those treated with sodium ascorbate. No significant difference was observed between the five-minute and 10-minute pretreatment times in groups II and III. It can be concluded that dentin surface pretreatment with both 10% sodium ascorbate and 6.5% proanthocyanidin resulted in significant improvement in bond strength of resin composite to deep dentin.

Aloe vera: an in vitro study of effects on corneal wound closure and collagenase activity.

Science.gov (United States)

Curto, Elizabeth M; Labelle, Amber; Chandler, Heather L

2014-11-01

To evaluate the in vitro effects of an aloe vera solution on (i) the viability and wound healing response of corneal cells and (ii) the ability to alter collagenase and gelatinase activities. Primary cultures of corneal epithelial cells and fibroblasts were prepared from grossly normal enucleated canine globes and treated with an aloe solution (doses ranging from 0.0-2 mg/mL). Cellular viability was evaluated using a colorimetric assay. A corneal wound healing model was used to quantify cellular ingrowth across a defect made on the confluent surface. Anticollagenase and antigelatinase activities were evaluated by incubating a bacterial collagenase/gelatinase with aloe solution (doses ranging from 0.0-500 μg/mL) and comparing outcome measures to a general metalloproteinase inhibitor, 1, 10-phenanthroline, and canine serum (doses ranging from 0.0-100%). None of the concentrations of aloe solution tested significantly affected the viability of corneal epithelial cells or fibroblasts. Concentrations ≤175 μg/mL slightly accelerated corneal epithelial cell wound closure; this change was not significant. Concentrations ≥175 μg/mL significantly (P ≤ 0.001) slowed the rate of corneal fibroblast wound closure, while aloe concentrations Aloe solution did not alter the ability for collagenase to degrade gelatin or collagen Type I but increased the ability for collagenase to degrade Type IV collagen. Although additional experiments are required, lower concentrations of aloe solution may be beneficial in healing of superficial corneal wounds to help decrease fibrosis and speed epithelialization. An increase in collagenase activity with aloe vera warrants further testing before considering in vivo studies. © 2014 American College of Veterinary Ophthalmologists.

The carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen in serum as a marker of bone resorption

DEFF Research Database (Denmark)

Hassager, C; Jensen, L T; Pødenphant, J

1994-01-01

Carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP) in serum has recently been proposed as a new biochemical marker of bone resorption. In the present study we compared serum ICTP with radiopharmaceutical and histomorphometric measurements of bone turnover...... in postmenopausal women with mild osteoporosis, and assessed the effect of hormone replacement therapy (HRT) (2 mg 17 beta-estradiol plus 1 mg norethisterone daily) and anabolic steroid therapy (50 mg nandrolone decanoate (ND) i.m. every 3 weeks) on serum ICTP in two double-blind placebo-controlled studies with 55...... to 75-year-old women. Serum ICTP measured by radioimmunoassay (RIA) correlated significantly with the 24-hour whole body retention of 99m-technetium diphosphonate (Rho = 0.47, P

The effect of vitamin C deficiency and chronic ultraviolet-B exposure on corneal ultrastructure: a preliminary investigation

Science.gov (United States)

Hayes, Sally; Cafaro, Thamara A.; Boguslawska, Patrycja J.; Kamma-Lorger, Christina S.; Boote, Craig; Harris, Jonathan; Young, Robert; Hiller, Jennifer; Terrill, Nicholas; Meek, Keith M.

2011-01-01

Purpose In the visually debilitating condition of climatic droplet keratopathy, corneal transparency is progressively lost. Although the precise cause of the disease and the mechanism by which it progresses are not known, a lifetime exposure to high solar radiation and a vitamin C–deficient diet may be involved in its development. This study examines the effect of dietary ascorbate levels and ultraviolet (UV)-B exposure on corneal stromal structure. Methods Eight guinea pigs were divided into four treatment groups (A, B, C, and D). For 15 weeks, Groups A and C were fed an ascorbate-rich diet (2 mg/100 g bodyweight/day), while Groups B and D received an ascorbate-deficient diet (0.07 mg/100 g bodyweight/day). For the last 12 weeks of the study, Groups C and D also experienced chronic UVB exposure (0.12 J/cm2 for 40 min/day). Following euthanasia, the corneas were enucleated and their stromal ultrastructure examined using X-ray scattering and electron microscopy. Results UVB exposure resulted in an increased corneal thickness (p<0.001), but this was not accompanied by a widespread expansion of the collagen fibrillar array, and in the case of ascorbate-deficient animals, stromal thickening was associated with the compaction of collagen fibrils (p<0.01). Neither UVB exposure nor ascorbic acid deficiency caused any change in the average diameter or D-periodicity of the stromal collagen fibrils. Conclusions UVB-induced changes in the corneal ultrastructure were most pronounced in animals fed an ascorbic acid–deficient diet. This suggests that ascorbic acid may play a vital role in protecting the corneal stroma from the harmful effects of UVB. PMID:22171156

Surface modification of electrospun PLGA scaffold with collagen for bioengineered skin substitutes

Energy Technology Data Exchange (ETDEWEB)

Sadeghi, A.R., E-mail: sadeghi_av@ymail.com [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Nokhasteh, S. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Molavi, A.M. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Materials Engineering Department, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Khorsand-Ghayeni, M. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Naderi-Meshkin, H. [Stem Cell and Regenerative Medicine Research Department, Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Mahdizadeh, A. [Nanotechnology Institute, University of Sistan and Baluchestan, Zahedan (Iran, Islamic Republic of)

2016-09-01

In skin tissue engineering, surface feature of the scaffolds plays an important role in cell adhesion and proliferation. In this study, non-woven fibrous substrate based on poly (lactic-co-glycolic acid) (PLGA) (75/25) were hydrolyzed in various concentrations of NaOH (0.05 N, 0.1 N, 0.3 N) to increase carboxyl and hydroxyl groups on the fiber surfaces. These functional groups were activated by EDC/NHS to create chemical bonding with collagen. To improve bioactivity, the activated substrates were coated with a collagen solution (2 mg/ml) and cross-linking was carried out using the EDC/NHS in MES buffer. The effectiveness of the method was evaluated by contact angle measurements, porosimetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile and degradation tests as well as in vitro cell attachment and cytotoxicity assays. Cell culture results of human dermal fibroblasts (HDF) and keratinocytes cell line (HaCat) revealed that the cells could attach to the scaffold. Further investigation with MTT assay showed that the cell proliferation of HaCat significantly increases with collagen coating. It seems that sufficient stability of collagen on the surface due to proper chemical bonding and cross-linking has increased the bioactivity of surface remarkably which can be promising for bioengineered skin applications. - Highlights: • Surface activation was carried out by hydrolysis of PLGA fibers. • To improve bioactivity, the activated samples were coated with a collagen solution. • Functional groups were activated by EDC/NHS to create chemical bonding with collagen. • Cross-linking of collagen was carried out using EDC/NHS in MES buffer. • The coated samples exhibited better adhesion and proliferation of epidermal cells.

Surface modification of electrospun PLGA scaffold with collagen for bioengineered skin substitutes

International Nuclear Information System (INIS)

Sadeghi, A.R.; Nokhasteh, S.; Molavi, A.M.; Khorsand-Ghayeni, M.; Naderi-Meshkin, H.; Mahdizadeh, A.

2016-01-01

In skin tissue engineering, surface feature of the scaffolds plays an important role in cell adhesion and proliferation. In this study, non-woven fibrous substrate based on poly (lactic-co-glycolic acid) (PLGA) (75/25) were hydrolyzed in various concentrations of NaOH (0.05 N, 0.1 N, 0.3 N) to increase carboxyl and hydroxyl groups on the fiber surfaces. These functional groups were activated by EDC/NHS to create chemical bonding with collagen. To improve bioactivity, the activated substrates were coated with a collagen solution (2 mg/ml) and cross-linking was carried out using the EDC/NHS in MES buffer. The effectiveness of the method was evaluated by contact angle measurements, porosimetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile and degradation tests as well as in vitro cell attachment and cytotoxicity assays. Cell culture results of human dermal fibroblasts (HDF) and keratinocytes cell line (HaCat) revealed that the cells could attach to the scaffold. Further investigation with MTT assay showed that the cell proliferation of HaCat significantly increases with collagen coating. It seems that sufficient stability of collagen on the surface due to proper chemical bonding and cross-linking has increased the bioactivity of surface remarkably which can be promising for bioengineered skin applications. - Highlights: • Surface activation was carried out by hydrolysis of PLGA fibers. • To improve bioactivity, the activated samples were coated with a collagen solution. • Functional groups were activated by EDC/NHS to create chemical bonding with collagen. • Cross-linking of collagen was carried out using EDC/NHS in MES buffer. • The coated samples exhibited better adhesion and proliferation of epidermal cells.

Chemical functionalization and stabilization of type I collagen with organic tanning agents

International Nuclear Information System (INIS)

Albu, Madalina Georgiana; Deselnicu, Viorica; Ioannidis, Ioannis; Deselnicu, Dana; Chelaru, Ciprian

2015-01-01

We investigated the interactions between selected organic tanning agents and type I fibrillar collagen as a model fibrillar substrate to enable the fast direct evaluation and validation of interpretations of tanning activity. Type I fibrillar collagen (1%) as gel was used as substrate of tanning and tannic acid, resorcinol- and melamine-formaldehyde and their combination at three concentrations as crosslinking agents (tannins). To evaluate the stability of collagen during tanning, the crosslinked gels at 2.8, 4.5 and 9.0 pHs were freeze-dried as discs which were characterized by FTIR, shrinkage temperature, enzymatic degradation and optical microscopy, and the results were validated by statistical analyses. The best stability was given by combinations between resorcinol- and melamine-formaldehyde at isoelectric pH

Chemical functionalization and stabilization of type I collagen with organic tanning agents

Energy Technology Data Exchange (ETDEWEB)

Albu, Madalina Georgiana; Deselnicu, Viorica; Ioannidis, Ioannis; Deselnicu, Dana; Chelaru, Ciprian [Leather and Footwear Research Institute, Bucharest (Romania)

2015-02-15

We investigated the interactions between selected organic tanning agents and type I fibrillar collagen as a model fibrillar substrate to enable the fast direct evaluation and validation of interpretations of tanning activity. Type I fibrillar collagen (1%) as gel was used as substrate of tanning and tannic acid, resorcinol- and melamine-formaldehyde and their combination at three concentrations as crosslinking agents (tannins). To evaluate the stability of collagen during tanning, the crosslinked gels at 2.8, 4.5 and 9.0 pHs were freeze-dried as discs which were characterized by FTIR, shrinkage temperature, enzymatic degradation and optical microscopy, and the results were validated by statistical analyses. The best stability was given by combinations between resorcinol- and melamine-formaldehyde at isoelectric pH.

Cross-linking for microbial keratitis

Directory of Open Access Journals (Sweden)

Jayesh Vazirani

2013-01-01

Full Text Available The success of collagen cross-linking as a clinical modality to modify the clinical course in keratoconus seems to have fueled the search for alternative applications for this treatment. Current clinical data on its efficacy is limited and laboratory data seems to indicate that it performs poorly against resistant strains of bacteria and against slow growing organisms. However, the biological plausibility of crosslinking and the lack of effective strategies in managing infections with these organisms continue to focus attention on this potential treatment. Well-conducted experimental and clinical studies with controls are required to answer the questions of its efficacy in future.

Photophysics and photochemistry of Riboflavin-induced corneal Cross-Linking

DEFF Research Database (Denmark)

Breitenbach, Thomas

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 show the contribution of singlet molecular oxygen O2(a1Δg) to this phenomena and show...

Collagen and mineral deposition in rabbit cortical bone during maturation and growth: effects on tissue properties.

Science.gov (United States)

Isaksson, Hanna; Harjula, Terhi; Koistinen, Arto; Iivarinen, Jarkko; Seppänen, Kari; Arokoski, Jari P A; Brama, Pieter A; Jurvelin, Jukka S; Helminen, Heikki J

2010-12-01

We characterized the composition and mechanical properties of cortical bone during maturation and growth and in adult life in the rabbit. We hypothesized that the collagen network develops earlier than the mineralized matrix. Growth was monitored, and the rabbits were euthanized at birth (newborn), and at 1, 3, 6, 9, and 18 months of age. The collagen network was assessed biochemically (collagen content, enzymatic and non-enzymatic cross-links) in specimens from the mid-diaphysis of the tibia and femur and biomechanically (tensile testing) from decalcified whole tibia specimens. The mineralized matrix was analyzed using pQCT and 3-point bend tests from intact femur specimens. The collagen content and the Young's modulus of the collagen matrix increased significantly until the rabbits were 3 months old, and thereafter remained stable. The amount of HP and LP collagen cross-links increased continuously from newborn to 18 months of age, whereas PEN cross-links increased after 6 months of age. Bone mineral density and the Young's modulus of the mineralized bone increased until the rabbits were at least 6 months old. We concluded that substantial changes take place during the normal process of development in both the biochemical and biomechanical properties of rabbit cortical bone. In cortical bone, the collagen network reaches its mature composition and mechanical strength prior to the mineralized matrix. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The evaluation of collagen gel with various connection states by using MRI

International Nuclear Information System (INIS)

Kudo, Hiroki; Mukai, Naoki; Gouping, Chen; Numanno, Tomokazu; Honma, Kazuhiro; Tateishi, Tetsuya; Miyanaga, Yutaka; Miyakawa, Syumpei

2008-01-01

To noninvasively evaluate the connection states of collagen fiber, a characterizing factor of the physical property, is considered to be helpful in the evaluation of cartilage functions. The purpose of this study was to examine how the connection states of collagen influence the MRI parameters by evaluating the collagen gel with various connection states using MRI. MRI was performed to six type I collagen gel samples with various connection status and a water sample. The evaluation parameters included T1 relaxation time, T2 relaxation time, and diffusion coefficient. With regard to gel samples with cross-links, the T2 relaxation time was shortened in proportion to the dose of glutaraldehyde. It is considered that as the glutaraldehyde concentration increases, the distance between protons in water molecules decreases; this is followed by a stronger bipole-bipole interaction, resulting in a shorter T2 relaxation time. The diffusion coefficient for gel samples with cross-links also decreased with increasing glutaraldehyde concentrations. However, gel samples without glutaraldehyde were almost the same as that of the water. This result suggested that the degree of entrapment of water inside the gel samples without cross-links, even when it converted into gel, was found to be nearly equal to that of the free water

Biohybrid Fibro-Porous Vascular Scaffolds: Effect of Crosslinking on Properties

OpenAIRE

Thomas, Vinoy; Nozik, Danna; Patel, Harsh; Singh, Raj K.; Vohra, Yogesh K.

2015-01-01

Tubular grafts were fabricated from blends of polycaprolactone (PCL) and poly(glycolide -co-caprolactone) (PGC) polymers and coated with an extracellular matrix containing collagens, laminin, and proteoglycans, but not growth factors (HuBiogel™). Multifunctional scaffolds from polymer blends and membrane proteins provide the necessary biomechanics and biological functions for tissue regeneration. Two crosslinking agents, a natural crosslinker namely genipin (Gp) and a carbodiimide reagent nam...

Collagen derived serum markers in carcinoma of the prostate

DEFF Research Database (Denmark)

Rudnicki, M; Jensen, L T; Iversen, P

1995-01-01

Three new collagen markers deriving from the collagenous matrix, e.g. carboxyterminal propeptide of type I procollagen (PICP), carboxy-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP), and aminoterminal propeptide of type III procollagen (PIIINP) were used for the diagnose...... of prostatic bone metastases. Blood samples were obtained prior to biopsy or TURP. Serum PICP, PIIINP and ICTP were measured with commercial available RIAs and PSA by IRMA. Serum PSA was increased in patients with local prostatic cancer compared with patients with hyperplasia (p

Mimicking corneal stroma using keratocyte-loaded photopolymerizable methacrylated gelatin hydrogels.

Science.gov (United States)

Kilic Bektas, Cemile; Hasirci, Vasif

2018-04-01

Cell-laden methacrylated gelatin (GelMA) hydrogels with high (approximately 90%) transparency were prepared to mimic the natural form and function of corneal stroma. They were synthesized from GelMA with a methacrylation degree of 70% as determined by nuclear magnetic resonance. Hydrogels were strong enough to withstand handling. Stability studies showed that 87% of the GelMA hydrogels remained after 21 days in phosphate buffered saline (PBS). Cell viability in the first 2 days was over 90% for the human keratocytes loaded in the gels as determined with the live-dead analysis. Cells in the hydrogel elongated and connected to each other as observed by confocal laser scanning microscopy (CLSM) images and scanning electron microscope analysis after 3 weeks in the culture medium and cells were seen to be distributed throughout the hydrogel bulk. Cells were found to synthesize collagen Types I and V, decorin, and biglycan (representative collagens and proteoglycans of human corneal stroma, respectively) showing that keratocytes maintained their functions and preserved their phenotypes in the hydrogels. Transparency of cell-loaded and cell-free hydrogels after 21 days was found to be over 90% at all time points in the visible light range and was comparable to the transparency of the native cornea. The corneal stroma equivalent produced in this study that has cells entrapped in it leads to a product with homogenous distribution of cells. It was transparent at the very beginning and is expected to allow better vision than nontransparent substrates. It, therefore, has a significant potential to be used as an alternative to the current products used to treat corneal blindness. Copyright © 2017 John Wiley & Sons, Ltd.

Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.

Science.gov (United States)

Hatou, Shin; Higa, Kazunari; Inagaki, Emi; Yoshida, Satoru; Kimura, Erika; Hayashi, Ryuhei; Tsujikawa, Motokazu; Tsubota, Kazuo; Nishida, Kohji; Shimmura, Shigeto

2013-12-01

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

Enhanced physicochemical properties of collagen by using EDC ...

Indian Academy of Sciences (India)

MS received 4 July 2011; revised 21 December 2011. Abstract. Collagen-based .... On the other hand, in the process of crosslinking, carboxyl and amino groups that .... logy Plan Project of Fujian Department of Education, China. (Grant no.

Glutaraldehyde Cross-Linking of TendonMechanical Effects at the Level of the Tendon Fascicle and Fibril

DEFF Research Database (Denmark)

Hansen, P.; Svensson, R.B.; Aagaard, P.

2009-01-01

were examined by atomic force microscopy. Peak forces increased from 1379 to 2622 pN while an extended Hertz fit of force-indentation data showed a 24 fold increase in Young's modulus on indentation. The effect of glutaraldehyde cross-linking on the tensile properties of a single collagen fibril......Conclusive insight into the microscopic principles that govern the strength of tendon and related connective tissues is lacking and the importance of collagen cross-linking has not been firmly established. The combined application of whole-tissue mechanical testing and atomic force spectroscopy...... allowed for a detailed characterization of the effect of cross-linking in rat-tail tendon. The cross-link inducing agent glutaraldehyde augmented the tensile strength of tendon fascicles. Stress at failure increased from 8 MPa to 39 MPa. The mechanical effects of glutaraldehyde at the tendon fibril level...

A two-year's results of iontophoresis-assisted transepithelial corneal cross-linking for progressive keratoconus

Directory of Open Access Journals (Sweden)

Hong-Zhen Jia

2016-07-01

Full Text Available AIM: To report a two-year's results of iontophoresis-assisted transepithelial corneal cross-linking(I-CXLfor progressive keratoconus. METHODS: Thirty-four eyes in 24 patients with progressive keratoconus(mean age 21.0±5.6 years; range: 14-32 yearswere treated. After 1g/L riboflavin-distilled water solution was administered by iontophoresis-assited(current 1mAtransepithelial method for 5min in total, standard surface UVA irradiation(370nm, 3mW/cm2was performed at a 1-cm distance for 30min. The best corrected visual acuity(BCVAmeasured as LogMAR number, corneal refractive astigmatism, K1, K2, Kmean, Kmax, intraocular pressure, endothelial cell density, the thickness at corneal apex and the thinnest point were measured preoperatively and 2a postoperatively. RESULTS:At 2a after the procedure, BCVA(LogMARimproved from 0.32±0.25 to 0.25±0.19(t=2.849, P=0.015. K1 decreased from 47.12±4.33 to 46.06±4.77(t=2.652, P=0.015. K2 decreased from 51.36±5.59 to 50.40±6.16(t=2.121, P=0.047. Kmean decreased from 49.12±4.76 to 48.10±5.25(t=2.663, P=0.015. Kmax decreased from 57.57±8.30 to 55.91±8.14(t=2.398, P=0.026. The corneal apex thickness decreased from 476.90±38.71μm to 454.43±40.86μm(t=2.853, P=0.010. The thinnest thickness decreased from 464.38±39.92μm to 433.86±50.78μm(t=3.485, P=0.002. Corneal refractive astigmatism, intraocular pressure and endothelial cell density did not show significant changes. CONCLUSION: I-CXL for progressive keratoconus is safe and effective which can prevent deterioration of progressive keratoconus within 2a, but further long-term studies are necessary still.

Low temperature corneal laser welding investigated by atomic force microscopy

Science.gov (United States)

Matteini, Paolo; Sbrana, Francesca; Tiribilli, Bruno; Pini, Roberto

2009-02-01

The structural modifications in the stromal matrix induced by low-temperature corneal laser welding were investigated by atomic force microscopy (AFM). This procedure consists of staining the wound with Indocyanine Green (ICG), followed by irradiation with a near-infrared laser operated at low-power densities. This induces a local heating in the 55-65 °C range. In welded tissue, extracellular components undergo heat-induced structural modifications, resulting in a joining effect between the cut edges. However, the exact mechanism generating the welding, to date, is not completely understood. Full-thickness cuts, 3.5 mm in length, were made in fresh porcine cornea samples, and these were then subjected to laser welding operated at 16.7 W/cm2 power density. AFM imaging was performed on resin-embedded semi-thin slices once they had been cleared by chemical etching, in order to expose the stromal bulk of the tissue within the section. We then carried out a morphological analysis of characteristic fibrillar features in the laser-treated and control samples. AFM images of control stromal regions highlighted well-organized collagen fibrils (36.2 +/- 8.7 nm in size) running parallel to each other as in a typical lamellar domain. The fibrils exhibited a beaded pattern with a 22-39 nm axial periodicity. Laser-treated corneal regions were characterized by a significant disorganization of the intralamellar architecture. At the weld site, groups of interwoven fibrils joined the cut edges, showing structural properties that were fully comparable with those of control regions. This suggested that fibrillar collagen is not denatured by low-temperature laser welding, confirming previous transmission electron microscopy (TEM) observations, and thus it is probably not involved in the closure mechanism of corneal cuts. The loss of fibrillar organization may be related to some structural modifications in some interfibrillar substance as proteoglycans or collagen VI. Furthermore, AFM

Biohybrid Fibro-Porous Vascular Scaffolds: Effect of Crosslinking on Properties.

Science.gov (United States)

Thomas, Vinoy; Nozik, Danna; Patel, Harsh; Singh, Raj K; Vohra, Yogesh K

Tubular grafts were fabricated from blends of polycaprolactone (PCL) and poly(glycolide -co-caprolactone) (PGC) polymers and coated with an extracellular matrix containing collagens, laminin, and proteoglycans, but not growth factors (HuBiogel™). Multifunctional scaffolds from polymer blends and membrane proteins provide the necessary biomechanics and biological functions for tissue regeneration. Two crosslinking agents, a natural crosslinker namely genipin (Gp) and a carbodiimide reagent namely 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), were used for further stabilizing the protein matrix and the effect of crosslinking was evaluated for structural, morphological, mechanical properties using SEM, DSC and DMA. SEM images and fiber diameter distribution showed fiber-size between 0.2 µm to 1 µm with the majority of fiber diameters being under 500 nm, indicating upper range of protein fiber-sizes (for example, collagen fibers in extracellular matrix are in 50 to 500 nm diameter range). HB coating did not affect the mechanical properties, but increased its hydrophilicity of the graft. Overall data showed that PCL/PGC blends with 3:1 mass ratio exhibited mechanical properties comparable to those of human native arteries (tensile strength of 1-2 MPa and Young's modulus of crosslinking on coating stability was investigated to assure the retention of proteins on scaffold for effective cell-matrix interactions.

Agar/collagen membrane as skin dressing for wounds

Energy Technology Data Exchange (ETDEWEB)

Bao Lei; Yang Wei; Mao Xuan; Mou Shansong; Tang Shunqing [Biomedical Engineering Institute, Jinan University, Guangzhou (China)], E-mail: tshunqt@jnu.edu.cn, E-mail: tmuss@jnu.edu.cn

2008-12-15

Agar, a highly hydrophilic polymer, has a special gel property and favorable biocompatibility, but moderate intension strength in an aqueous condition and a low degradation rate. In order to tailor both properties of mechanical intension and degradation, type I collagen was composited with agar in a certain ratio by drying at 50 {sup 0}C or by a freeze-dry process. Glutaraldehyde was chosen as a crosslinking agent, and the most favorable condition for crosslinking was that the weight ratio of agar to glutaraldehyde was 66.7 and the pH value about 5. Dynamic mechanical analysis results showed that the single agar membrane had a modulus value between 640 MPa and 1064 MPa, but it was between 340 MPa and 819 MPa after being composited with type I collagen. It was discovered under an optical microscope that the pores were interconnected in the composite scaffolds instead of the honeycomb-like pores in a single type I collagen scaffold or the laminated gaps in a single agar scaffold. The results of an acute toxicity test disclosed that the composites were not toxic to mice although the composites were crosslinked with a certain concentration of glutaraldehyde. The results of gross examinations showed that when the composite membranes or scaffolds were applied to a repair rabbit skin lesion, the composites had a good repair effect without infection, liquid exudation or visible scar in the lesion covered with them. But in the control group, the autologous skin showed necrosis and there were a lot of scar tissues in the lesion site. H and E staining results showed that the repair tissue was similar to the normal one and very few scaffolds or membranes were left without degradation after 2 or 3 weeks. In conclusion, it is proved that type I collagen increases the toughness of the agar membrane, and the agar/type I collagen composites are promising biomaterials as wound dressings for healing burns or ulcers.

Agar/collagen membrane as skin dressing for wounds

International Nuclear Information System (INIS)

Bao Lei; Yang Wei; Mao Xuan; Mou Shansong; Tang Shunqing

2008-01-01

Agar, a highly hydrophilic polymer, has a special gel property and favorable biocompatibility, but moderate intension strength in an aqueous condition and a low degradation rate. In order to tailor both properties of mechanical intension and degradation, type I collagen was composited with agar in a certain ratio by drying at 50 0 C or by a freeze-dry process. Glutaraldehyde was chosen as a crosslinking agent, and the most favorable condition for crosslinking was that the weight ratio of agar to glutaraldehyde was 66.7 and the pH value about 5. Dynamic mechanical analysis results showed that the single agar membrane had a modulus value between 640 MPa and 1064 MPa, but it was between 340 MPa and 819 MPa after being composited with type I collagen. It was discovered under an optical microscope that the pores were interconnected in the composite scaffolds instead of the honeycomb-like pores in a single type I collagen scaffold or the laminated gaps in a single agar scaffold. The results of an acute toxicity test disclosed that the composites were not toxic to mice although the composites were crosslinked with a certain concentration of glutaraldehyde. The results of gross examinations showed that when the composite membranes or scaffolds were applied to a repair rabbit skin lesion, the composites had a good repair effect without infection, liquid exudation or visible scar in the lesion covered with them. But in the control group, the autologous skin showed necrosis and there were a lot of scar tissues in the lesion site. H and E staining results showed that the repair tissue was similar to the normal one and very few scaffolds or membranes were left without degradation after 2 or 3 weeks. In conclusion, it is proved that type I collagen increases the toughness of the agar membrane, and the agar/type I collagen composites are promising biomaterials as wound dressings for healing burns or ulcers.

Multiple molecular forms of pyridinoline crosslinks generated by the ...

African Journals Online (AJOL)

molecular forms of pyridinoline crosslinks from insoluble bone type I collagen, and we studied ... resorbed in a dynamic process during ... proteins at 4 C for 24H in 4 mol/L .... fragments was merely due to interactions ... completely elucidated.

Vision Restoration with a Collagen Crosslinked Boston Keratoprosthesis Unit

Science.gov (United States)

2016-09-01

ex vivo using vitamin B2 (riboflavin) and ultraviolet light. The overall objective of this study is to prevent sight-threatening keratoprosthesis...keratoprosthesis carrier cornea using tissue that has been cross-linked using vitamin B2 (riboflavin) and ultraviolet light prior to prosthesis

Effects of alginate hydrogel cross-linking density on mechanical and biological behaviors for tissue engineering.

Science.gov (United States)

Jang, Jinah; Seol, Young-Joon; Kim, Hyeon Ji; Kundu, Joydip; Kim, Sung Won; Cho, Dong-Woo

2014-09-01

An effective cross-linking of alginate gel was made through reaction with calcium carbonate (CaCO3). We used human chondrocytes as a model cell to study the effects of cross-linking density. Three different pore size ranges of cross-linked alginate hydrogels were fabricated. The morphological, mechanical, and rheological properties of various alginate hydrogels were characterized and responses of biosynthesis of cells encapsulated in each gel to the variation in cross-linking density were investigated. Desired outer shape of structure was maintained when the alginate solution was cross-linked with the applied method. The properties of alginate hydrogel could be tailored through applying various concentrations of CaCO3. The rate of synthesized GAGs and collagens was significantly higher in human chondrocytes encapsulated in the smaller pore structure than that in the larger pore structure. The expression of chondrogenic markers, including collagen type II and aggrecan, was enhanced in the smaller pore structure. It was found that proper structural morphology is a critical factor to enhance the performance and tissue regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of photoactivated riboflavin on the biodegradation-resistance of root-dentin collagen.

Science.gov (United States)

Priyadarshini, Balasankar Meera; Lu, Thong Beng; Fawzy, Amr S

2017-12-01

This study was conducted to evaluate the effect of UVA-activated 1% riboflavin solution on structural integrity; mechanical properties and stability; and collagenase-mediated collagen solubilisation resistance of demineralized root dentin collagen matrix. Root dentin specimens demineralized with 17% EDTA for 7days were treated with 1% RF for 1min followed by UVA photo-activation at intensity 7mW/cm 2 for 1min. Control specimens were completely devoid of riboflavin and/or UVA treatments. Specimens were challenged with bacterial collagenase type-I solution for different time-periods at 37°C. Collagen solubilisation resistance was evaluated in terms of hydroxyproline (HYP) liberation. Mechanical characterization of dentin specimens before and after 24h of exposure to collagenase solution was done in terms of apparent-elastic modulus (E appr ) and ultimate tensile strength (UTS). Variations in dentin collagen-network structure with exposure time in collagenase were visualized by TEM. Crosslinking dentin with UVA-activated riboflavin significantly decreased HYP release and increased E appr and UTS compared to control specimens with storage time in collagenase. Moreover, crosslinked specimens showed higher structural resistance to collagenase effect reflected from dense, well-formed collagen fibrils-network with characteristic collagen cross-banding. UVA-activated riboflavin treatment increased collagenase-mediated collagen degradation resistance and enhanced mechanical stability against collagenase challenges of root dentin after EDTA demineralization. Copyright © 2017 Elsevier B.V. All rights reserved.

The biochemical changes of bone collagen after high-dose irradiation

International Nuclear Information System (INIS)

Tajiri, Ken

1980-01-01

In our clinic, patients with malignant bone tumors have been treated by high-dose irradiation therapy, 10,000-20,000 rads, for primary lesions. In order to study the biochemical changes of normal bone around tumor tissue, especially bone collagen, after high-dose irradiation, the author performed the following experiments. The right knee joint of rabbits was irradiated with either 6,000, 10,000, or 15,000 rads by 60 Co-γ ray. The cortical bone of the right tibial metaphysis was used for analyses and compared with the left tibia of the same rabbit. These studies were followed for one year after the final irradiation. The calcium, phosphorous and collagen contents of irradiated bone were remarkably changed. These data indicate that collagen biosynthesis of irradiated bone was decreased and the calcification was disturbed. An increase in the amount of total soluble collagen and a decrease in the amount of hydroxylysine bound sugar were observed. The ratio of β to α chains of the collagen molecule was also changed by the irradiation. The amount of reducible cross-links per hydroxyproline residue was strikingly increased three months after the final irradiation. These changes were remarkable especially in the 10,000 and 15,000 rads irradiated group and found to be recovered approximately six months to one year after the final irradiation. These findings indicate that high-dose irradiation reduces the stability of bone collagen both with the destruction of sugar bonds of hydroxylysine residues and the replacement of matured collagen matrix to immatured one which contain mostly labile reducible cross-links. (author)

Fracture mechanics of collagen fibrils

DEFF Research Database (Denmark)

Svensson, Rene B; Mulder, Hindrik; Kovanen, Vuokko

2013-01-01

Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within...... fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy...... technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human...

Fluorescence studies on the aggregation behaviors of collagen modified with NHS-activated poly(γ-glutamic acid).

Science.gov (United States)

Zhang, Min; Yang, Junhui; Yang, Qili; Huang, Liulian; Wu, Hui; Chen, Lihui; Ding, Cuicui

2018-06-01

The poly(γ-glutamic acid)-NHS (γ-PGA-NHS) esters were used to endow collagen with both of excellent water-solubility and thermal stability via cross-linking reaction between γ-PGA-NHS and collagen. In the present work, the effect of γ-PGA-NHS on the aggregation of collagen molecules was studied by fluorescence techniques. The fluorescence emission spectra of pyrene in collagen solutions and the intrinsic fluorescence emission spectra of collagen suggested different effects of γ-PGA-NHS on collagen molecules: inhibiting aggregation below critical aggregation concentration (CAC) and promoting aggregation above CAC. The two-dimensional (2D) fluorescence correlation spectra indicated that the intermolecular hydrogen bonding and cross-linking between γ-PGA-NHS and collagen would influence the aggregation of collagen molecules. By the ultra-sensitive differential scanning calorimeter (VP-DSC), it was found that the main denaturational transition temperature (T m2 ) of modified collagen increased, while its calorimetric enthalpy changes (ΔH 2 ) decreased compared to those of native collagen, further indicating that the modification of γ-PGA-NHS influenced the aggregation of collagen molecules. The study provide useful information for the utilizing and or the processing of water-soluble collagen in aqueous solution in the fields such as cosmetics, health care products, tissue engineering and biomedical materials, etc. Copyright © 2018 Elsevier B.V. All rights reserved.

Diaphragm Repair with a Novel Cross-Linked Collagen Biomaterial in a Growing Rabbit Model.

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Steffi Mayer

Full Text Available Neonates with congenital diaphragmatic hernia and large defects often require patch closure. Acellular collagen matrices (ACM have been suggested as an alternative to synthetic durable patches as they are remodeled by the host or could also be used for tissue engineering purposes.2.0x1.0 cm diaphragmatic defects were created in 6-weeks old New-Zealand white rabbits. We compared reconstruction with a purpose-designed cross-linked ACM (Matricel to 4-layer non-cross-linked small intestinal submucosa (SIS and a 1-layer synthetic Dual Mesh (Gore-Tex. Unoperated animals or animals undergoing primary closure (4/0 polyglecaprone served as age-matched controls. 60 (n = 25 resp. 90 (n = 17 days later, animals underwent chest x-ray and obduction for gross examination of explants, scoring of adhesion and inflammatory response. Also, uniaxial tensiometry was done, comparing explants to contralateral native diaphragmatic tissue.Overall weight nearly doubled from 1,554±242 g at surgery to 2,837±265 g at obduction (+84%. X-rays did show rare elevation of the left diaphragm (SIS = 1, Gore-Tex = 1, unoperated control = 1, but no herniation of abdominal organs. 56% of SIS and 10% of Matricel patches degraded with visceral bulging in four (SIS = 3, Matricel = 1. Adhesion scores were limited: 0.5 (Matricel to 1 (SIS, Gore-Tex to the left lung (p = 0.008 and 2.5 (Gore-Tex, 3 (SIS and 4 (Matricel to the liver (p<0.0001. Tensiometry revealed a reduced bursting strength but normal compliance for SIS. Compliance was reduced in Matricel and Gore-Tex (p<0.01. Inflammatory response was characterized by a more polymorphonuclear cell (SIS resp. macrophage (Matricel type of infiltrate (p<0.05. Fibrosis was similar for all groups, except there was less mature collagen deposited to Gore-Tex implants (p<0.05.Matricel induced a macrophage-dominated inflammatory response, more adhesions, had appropriate strength but a lesser compliance compared to native tissue. The herein

Disorganized collagen scaffold interferes with fibroblast mediated deposition of organized extracellular matrix in vitro.

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Saeidi, Nima; Guo, Xiaoqing; Hutcheon, Audrey E K; Sander, Edward A; Bale, Shyam Sundar; Melotti, Suzanna A; Zieske, James D; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W

2012-10-01

Many tissue engineering applications require the remodeling of a degradable scaffold either in vitro or in situ. Although inefficient remodeling or failure to fully remodel the temporary matrix can result in a poor clinical outcome, very few investigations have examined in detail, the interaction of regenerative cells with temporary scaffoldings. In a recent series of investigations, randomly oriented collagen gels were directly implanted into human corneal pockets and followed for 24 months. The resulting remodeling response exhibited a high degree of variability which likely reflects differing regenerative/synthetic capacity across patients. Given this variability, we hypothesize that a disorganized, degradable provisional scaffold could be disruptive to a uniform, organized reconstruction of stromal matrix. In this investigation, two established corneal stroma tissue engineering culture systems (collagen scaffold-based and scaffold-free) were compared to determine if the presence of the disorganized collagen gel influenced matrix production and organizational control exerted by primary human corneal fibroblast cells (PHCFCs). PHCFCs were cultured on thin disorganized reconstituted collagen substrate (RCS--five donors: average age 34.4) or on a bare polycarbonate membrane (five donors: average age 32.4 controls). The organization and morphology of the two culture systems were compared over the long-term at 4, 8, and 11/12 weeks. Construct thickness and extracellular matrix organization/alignment was tracked optically with bright field and differential interference contrast (DIC) microscopy. The details of cell/matrix morphology and cell/matrix interaction were examined with standard transmission, cuprolinic blue and quick-freeze/deep-etch electron microscopy. Both the scaffold-free and the collagen-based scaffold cultures produced organized arrays of collagen fibrils. However, at all time points, the amount of organized cell-derived matrix in the scaffold

Effects of advanced glycation end-product inhibition and cross-link breakage in diabetic rats

DEFF Research Database (Denmark)

Oturai, P S; Christensen, M; Rolin, B

2000-01-01

), and a breaker of already formed AGE cross-links, N-phenacylthiazolium bromide (PTB), were investigated in streptozotocin-diabetic female Wistar rats. Diabetes for 24 weeks resulted in decreased tail collagen pepsin solubility, reflecting the formation of AGE cross-linking. Collagen solubility was significantly...... ameliorated by treatment with NNC39-0028, whereas PTB had no effect. Increased urinary albumin excretion (UAE) in diabetic rats was observed in serial measurements throughout the study period, and was not reduced by any treatment. Vascular dysfunction in the eye, measured as increased clearance of 125I......-albumin, was induced by diabetes. NNC39-0028 did not affect this abnormality. This study demonstrated a pharmacological inhibition of collagen solubility alterations in diabetic rats without affecting diabetes-induced pathophysiology such as the increase in UAE or albumin clearance. Treatment with PTB, a specific...

Can Riboflavin Penetrate Stroma Without Disrupting Integrity of Corneal Epithelium in Rabbits? Iontophoresis and Ultraperformance Liquid Chromatography With Electrospray Ionization Tandem Mass Spectrometry.

Science.gov (United States)

Novruzlu, Şahin; Türkcü, Ümmühani Özel; Kvrak, İbrahim; Kvrak, Şeyda; Yüksel, Erdem; Deniz, Nuriye Gökçen; Bilgihan, Ayşe; Bilgihan, Kamil

2015-08-01

To examine riboflavin concentrations in corneas and aqueous humor from rabbits with standard and transepithelial methods and iontophoresis without disrupting the integrity of the corneal epithelium before corneal collagen cross-linking. Twenty-four eyes of 12 adult New Zealand rabbits were used. They were assigned to 4 groups, each including 6 eyes. Group 1 was exposed to the standard method and given riboflavin 0.1% after epithelial debridement. Group 2 was exposed to the transepithelial method and given benzalkonium chloride (BAC), ethylenediaminetetraacetic acid (EDTA), trometamol (TRIS), hydroxypropylmethylcellulose (HPMC), and riboflavin 0.2% 3 times at 1.5-minute intervals followed by riboflavin 0.2%. Group 3 was given riboflavin 0.1% by using 1-mA electric current for 10 minutes with the help of iontophoresis without using substances disrupting the integrity of the corneal epithelium. Group 4 received the same treatment as did group 3, except that it was given riboflavin 0.2%. Following these treatments, riboflavin concentrations in aqueous humor and corneas were measured with ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Riboflavin concentrations in the cornea and aqueous humor were higher in group 1 (42.4 ± 5.4 μg/g) than in the other groups. They were significantly higher in group 4 (34.2 ± 6.6 μg/g) than in group 2 (24.4 ± 1.2 μg/g) (P = 0.009) and group 3 (23.6 ± 6.1 μg/g) (P = 0.026). There was not a significant difference in corneal riboflavin concentrations between group 2 and group 3 (P = 0.937). Intrastromal and aqueous riboflavin concentrations after administration of riboflavin 0.2% through iontophoresis without disrupting the integrity of the corneal epithelium were lower than those after the standard method, but higher than those after the transepithelial method. In this study, in which riboflavin concentrations were measured with a very sensitive method

Effects of edible bird's nest (EBN on cultured rabbit corneal keratocytes

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Hun Lee

2011-10-01

Full Text Available Abstract Background There has been no effective treatment or agent that is available for corneal injury in promoting corneal wound healing. Previous studies on edible bird's nest extract (EBN had reported the presence of hormone-like substance; avian epidermal growth factor that could stimulate cell division and enhance regeneration. This study aimed to investigate the effects of EBN on corneal keratocytes proliferative capacity and phenotypical changes. Methods Corneal keratocytes from six New Zealand White Rabbits were isolated and cultured until Passage 1. The proliferative effects of EBN on corneal keratocytes were determined by MTT assay in serum-containing medium (FDS and serum-free medium (FD. Keratocytes phenotypical changes were morphologically assessed and gene expression of aldehyde dehydrogenase (ALDH, collagen type 1 and lumican were determined through RT-PCR. Results The highest cell proliferation was observed when both media were supplemented with 0.05% and 0.1% EBN. Cell proliferation was also consistently higher in FDS compared to FD. Both phase contrast micrographs and gene expression analysis confirmed the corneal keratocytes retained their phenotypes with the addition of EBN. Conclusions These results suggested that low concentration of EBN could synergistically induce cell proliferation, especially in serum-containing medium. This could be a novel breakthrough as both cell proliferation and functional maintenance are important during corneal wound healing. The in vitro test is considered as a crucial first step for nutri-pharmaceutical formation of EBN-based eye drops before in vivo application.

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

Science.gov (United States)

Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K

2018-02-01

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils

Intrastromal corneal ring implants for corneal thinning disorders: an evidence-based analysis.

Science.gov (United States)

2009-01-01

progressively thins causing a cone-like bulge or forward protrusion in response to the normal pressure of the eye. Thinning occurs primarily in the stoma layers and is believed to be a breakdown in the collagen network. This bulging can lead to an irregular shape or astigmatism of the cornea and, because the anterior part of the cornea is largely responsible for the focusing of light on the retina, results in loss of visual acuity. This can make even simple daily tasks, such as driving, watching television or reading, difficult to perform. Keratoconus (KC) is the most common form of corneal thinning disorder and is a noninflammatory chronic disease process. Although the specific causes of the biomechanical alterations that occur in KC are unknown, there is a growing body of evidence to suggest that genetic factors may play an important role. KC is a rare condition (management for this condition follows a step-wise approach depending on disease severity. Contact lenses are the primary treatment of choice when there is irregular astigmatism associated with the disease. When patients can no longer tolerate contact lenses or when lenses no longer provide adequate vision, patients are referred for corneal transplant. Keratoconus is one of the leading indications for corneal transplants and has been so for the last three decades. Yet, despite high graft survival rates of up to 20 years, there are reasons to defer receiving transplants for as long as possible. Patients with keratoconus are generally young and life-long term graft survival would be an important consideration. The surgery itself involves lengthy time off work and there are potential complications from long term steroid use following surgery, as well as the risk of developing secondary cataracts, glaucoma etc. After transplant, recurrent KC is possible with need for subsequent intervention. Residual refractive errors and astigmatism can remain challenging after transplantation and high refractive surgery rates and re

The potential role of bioengineering and three-dimensional printing in curing global corneal blindness.

Science.gov (United States)

Ludwig, Parker E; Huff, Trevor J; Zuniga, Jorge M

2018-01-01

An insufficiency of accessible allograft tissue for corneal transplantation leaves many impaired by untreated corneal disease. There is promise in the field of regenerative medicine for the development of autologous corneal tissue grafts or collagen-based scaffolds. Another approach is to create a suitable corneal implant that meets the refractive needs of the cornea and is integrated into the surrounding tissue but does not attempt to perfectly mimic the native cornea on a cellular level. Materials that have been investigated for use in the latter concept include natural polymers such as gelatin, semisynthetic polymers like gelatin methacrylate, and synthetic polymers. There are advantages and disadvantages inherent in natural and synthetic polymers: natural polymers are generally more biodegradable and biocompatible, while synthetic polymers typically provide greater control over the characteristics or property adjustment of the materials. Additive manufacturing could aid in the precision production of keratoprostheses and the personalization of implants.

Fish collagen is an important panallergen in the Japanese population.

Science.gov (United States)

Kobayashi, Y; Akiyama, H; Huge, J; Kubota, H; Chikazawa, S; Satoh, T; Miyake, T; Uhara, H; Okuyama, R; Nakagawara, R; Aihara, M; Hamada-Sato, N

2016-05-01

Collagen was identified as a fish allergen in early 2000s. Although its allergenic potential has been suggested to be low, risks associated with collagen as a fish allergen have not been evaluated to a greater extent. In this study, we aimed to clarify the importance of collagen as a fish allergen. Our results showed that 50% of Japanese patients with fish allergy had immunoglobulin E (IgE) against mackerel collagen, whereas 44% had IgE against mackerel parvalbumin. IgE inhibition assay revealed high cross-reactivity of mackerel collagen to 22 fish species (inhibition rates: 87-98%). Furthermore, a recently developed allergy test demonstrated that collagen triggered IgE cross-linking on mast cells. These data indicate that fish collagen is an important and very common panallergen in fish consumed in Japan. The high rate of individuals' collagen allergy may be attributable to the traditional Japanese custom of raw fish consumption. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Keratoconus: current perspectives

Directory of Open Access Journals (Sweden)

Vazirani J

2013-10-01

Full Text Available Jayesh Vazirani, Sayan BasuCornea and Anterior Segment Services, LV Prasad Eye Institute, Hyderabad, IndiaAbstract: Keratoconus is characterized by progressive corneal protrusion and thinning, leading to irregular astigmatism and impairment in visual function. The etiology and pathogenesis of the condition are not fully understood. However, significant strides have been made in early clinical detection of the disease, as well as towards providing optimal optical and surgical correction for improving the quality of vision in affected patients. The past two decades, in particular, have seen exciting new developments promising to alter the natural history of keratoconus in a favorable way for the first time. This comprehensive review focuses on analyzing the role of advanced imaging techniques in the diagnosis and treatment of keratoconus and evaluating the evidence supporting or refuting the efficacy of therapeutic advances for keratoconus, such as newer contact lens designs, collagen crosslinking, deep anterior lamellar keratoplasty, intracorneal ring segments, photorefractive keratectomy, and phakic intraocular lenses.Keywords: keratoconus, corneal topography, hydrops, collagen cross-linking, keratoplasty, contact lenses

A dense and strong bonding collagen film for carbon/carbon composites

Energy Technology Data Exchange (ETDEWEB)

Cao, Sheng; Li, Hejun, E-mail: lihejun@nwpu.edu.cn; Li, Kezhi; Lu, Jinhua; Zhang, Leilei

2015-08-30

Graphical abstract: - Highlights: • Significantly enhancement of biocompatibility on C/C composites by preparing a collagen film. • The dense and continuous collagen film had a strong bonding strength with C/C composites after dehydrathermal treatment (DHT) crosslink. • Numerous oxygen-containing functional groups formed on the surface of C/C composites without matrix damage. - Abstract: A strong bonding collagen film was successfully prepared on carbon/carbon (C/C) composites. The surface conditions of the modified C/C composites were detected by contact angle measurements, scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and Raman spectra. The roughness, optical morphology, bonding strength and biocompatibility of collagen films at different pH values were detected by confocal laser scanning microscope (CLSM), universal test machine and cytology tests in vitro. After a 4-h modification in 30% H{sub 2}O{sub 2} solution at 100 °C, the contact angle on the surface of C/C composites was decreased from 92.3° to 65.3°. Large quantities of hydroxyl, carboxyl and carbonyl functional groups were formed on the surface of the modified C/C composites. Then a dense and continuous collagen film was prepared on the modified C/C substrate. Bonding strength between collagen film and C/C substrate was reached to 8 MPa level when the pH value of this collagen film was 2.5 after the preparing process. With 2-day dehydrathermal treatment (DHT) crosslinking at 105 °C, the bonding strength was increased to 12 MPa level. At last, the results of in vitro cytological test showed that this collagen film made a great improvement on the biocompatibility on C/C composites.

A dense and strong bonding collagen film for carbon/carbon composites

International Nuclear Information System (INIS)

Cao, Sheng; Li, Hejun; Li, Kezhi; Lu, Jinhua; Zhang, Leilei

2015-01-01

Graphical abstract: - Highlights: • Significantly enhancement of biocompatibility on C/C composites by preparing a collagen film. • The dense and continuous collagen film had a strong bonding strength with C/C composites after dehydrathermal treatment (DHT) crosslink. • Numerous oxygen-containing functional groups formed on the surface of C/C composites without matrix damage. - Abstract: A strong bonding collagen film was successfully prepared on carbon/carbon (C/C) composites. The surface conditions of the modified C/C composites were detected by contact angle measurements, scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS) and Raman spectra. The roughness, optical morphology, bonding strength and biocompatibility of collagen films at different pH values were detected by confocal laser scanning microscope (CLSM), universal test machine and cytology tests in vitro. After a 4-h modification in 30% H 2 O 2 solution at 100 °C, the contact angle on the surface of C/C composites was decreased from 92.3° to 65.3°. Large quantities of hydroxyl, carboxyl and carbonyl functional groups were formed on the surface of the modified C/C composites. Then a dense and continuous collagen film was prepared on the modified C/C substrate. Bonding strength between collagen film and C/C substrate was reached to 8 MPa level when the pH value of this collagen film was 2.5 after the preparing process. With 2-day dehydrathermal treatment (DHT) crosslinking at 105 °C, the bonding strength was increased to 12 MPa level. At last, the results of in vitro cytological test showed that this collagen film made a great improvement on the biocompatibility on C/C composites

Combined use of chondroitinase-ABC, TGF-β1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair.

Science.gov (United States)

Makris, Eleftherios A; MacBarb, Regina F; Paschos, Nikolaos K; Hu, Jerry C; Athanasiou, Kyriacos A

2014-08-01

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility. Copyright © 2014 Elsevier Ltd. All rights reserved.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

DEFF Research Database (Denmark)

González-Santamaría, José; Villalba, María; Busnadiego, Oscar

2016-01-01

arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross......-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS: Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI...... resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION: LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery....

Donor cross-linking for keratoplasty: a laboratory evaluation.

Science.gov (United States)

Mukherjee, Achyut; Hayes, Sally; Aslanides, Ioannis; Lanchares, Elena; Meek, Keith M

2015-12-01

This laboratory-based investigation compares the topographic outcomes of conventional penetrating keratoplasty with that of a novel procedure in which donor corneas are cross-linked prior to keratoplasty. Penetrating keratoplasty procedures with continuous running sutures were carried out in a porcine whole globe model. Sixty eyes were randomly paired as 'donor' and 'host' tissue before being assigned to one of two groups. In the cross-linked group, donor corneas underwent riboflavin/UVA cross-linking prior to being trephined and sutured to untreated hosts. In the conventional keratoplasty group, both host and donor corneas remained untreated prior to keratoplasty. Topographic and corneal wavefront measurements were performed following surgery, and technical aspects of the procedure evaluated. Mean keratometric astigmatism was significantly lower in the cross-linked donor group at 3.67D (SD 1.8 D), vs. 8.43 D (SD 2.4 D) in the conventional keratoplasty group (p < 0.005). Mean wavefront astigmatism was also significantly reduced in the cross-linked donor group 4.71 D (SD 2.1) vs. 8.29D (SD 3.6) in the conventional keratoplasty group (p < 0.005). Mean RMS higher order aberration was significantly lower in the cross-linked donor group at 1.79 um (SD 0.98), vs. 3.05 um (SD 1.9) in the conventional keratoplasty group (P = 0.02). Qualitative analysis revealed less tissue distortion at the graft-host junction in the cross-linked group. Cross-linking of donor corneas prior to keratoplasty reduces intraoperative induced astigmatism and aberrations in an animal model. Further studies are indicated to evaluate the implications of this potential modification of keratoplasty surgery.

Corneal surface reconstruction - a short review

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Madhavan H N

2009-01-01

Full Text Available Cornea is the clear, dome-shaped surface that covers the front of the eye and when damage due to burns or injury and several other diseases, stem cells residing in its rim called "limbus" are stimulated to multiply to support growth of new epithelial cells over its surface. If this ready source of stem cells is damaged or destroyed the natural repair is not possible and such a condition is known as corneal limbal stem cell deficiency (CLSCD disease. Stem cell transplant helps such persons to regenerate the corneal surface. Human corneal limbal stem cell transplantation is at present an established procedure with reasonable good clinical outcome particularly when autologous limbal epithelial tissue from a fellow unaffected eye is used. 1, 2 A major concern related to the autograft is the possibility of CLSCD at the donor site, 3 techniques that allowed the expansion of a small limbal biopsy in the laboratory using cell cultures that could be then transplanted to the affected eye have been developed ,4, 5 Human amniotic membrane (HAM is used as a scaffold for both culturing the human limbal epithelial cells and for ocular surface reconstruction with the cultured limbal epithelial cells. 4-7 However, researchers have used alternative scaffolds like collagen 8, fibrin gel 9 and cross-linked gel of fibronectin and fibrin. 10 All these are biological materials and also need for animal 3T3 feeder layer for stem cell cultures. The properties of HAM are unique including antiadhesive effects, bacteriostatic effects, wound protection, pain reduction, and improvement of epithelialization and characteristically lacking imunogenicity. The use of amniotic membrane transplantation (AMT to treat ocular surface abnormalities was first reported by Graziella Pellegrini, chief of stem cell laboratory at Giovanni Paolo Hospital in Venice, Italy, who was the first to demonstrate the limbal stem cell transplant in 1997. Amniotic membrane has been successfully used in

Higher order optical aberrations and visual acuity in a randomized controlled trial comparing transepithelial versus epithelium-off corneal crosslinking for progressive keratoconus

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Godefrooij DA

2017-10-01

Full Text Available Daniel A Godefrooij, Mustapha El Kandoussi, Nienke Soeters, Robert PL Wisse Utrecht Cornea Research Group, Department of Ophthalmology, University Medical Center Utrecht, Utrecht, the Netherlands Purpose: The purpose of this study was to compare the effects of transepithelial crosslinking (trans-CXL versus epithelium-off crosslinking (epi-off CXL for progressive keratoconus with respect to the development of higher order aberrations (HOAs and their effects on visual acuity.Materials and methods: A total of 61 patients were randomized and examined preoperatively and 1, 3, 6, and 12 months postoperatively in an academic referral center. Total corneal HOAs were compared between the two treatment groups using mixed linear modeling. Types of HOAs (coma, trefoil, and spherical aberration that differed between groups were entered in a multivariable analysis to test their effect on uncorrected distance visual acuity (UDVA and corrected distance visual acuity (CDVA.Results: The epi-off CXL group had more flattening in maximal keratometry compared to the trans-CXL group (P=0.02. UDVA did not differ significantly between the groups (P=0.59; however, CDVA was significantly more improved in the trans-CXL group (P=0.02. Horizontal trefoil improved more in the epi-off group compared to the trans-CXL group (P=0.04, whereas the other HOAs were virtually unchanged in both groups. Differences in changes in HOAs between the two groups had no effect on either UCVA (P=0.76 or CDVA (P=0.96.Conclusion: Although HOAs are clinically relevant determinants of vision quality in keratoconus patients, the change in total HOAs post treatment did not differ between the trans-CXL and epi-off CXL groups. Only horizontal trefoil differed significantly post treatment between the trans-CXL and epi-off CXL groups. However, this difference did not independently affect either UDVA or CDVA. Trans-CXL provides no benefit over epi-off CXL regarding visual relevant HOAs. Keywords

Are the surgeons safe during UV-A radiation exposure in collagen cross-linking procedure?

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Shetty, Rashmi; Shetty, Rohit; Mahendradas, Padmamalini; Shetty, Bhujang K

2012-02-01

To quantify the effect of scattered UV-A radiation used in the collagen cross-linking (CXL) procedure and the amount of radiation reaching the surgeon and the surrounding area and to estimate the dampening effect by various protective devices. In this case series, 3 patients [aged 25-30 (±2.5) years] with keratoconus underwent a CXL procedure with UV-A light and riboflavin. Irradiance was measured using a spectrometer (Model USB2000; Ocean Optics, Inc) for various distances from the source, at various angles, and for different durations of radiation. The spectrometer was also used to measure the dampening effect produced by gown, latex gloves, and UV-protective glasses. Maximum UV-A radiation (1.4 × 10(-9) mW/cm(2)) was measured at 2 cm from the limbus, when the probe was held at a 45-degree angle to the floor. UV-A radiation reaching the surgeon's eye and the abdomen was 3.403 × 10(-11) and 2.36 × 10(-11) mW/cm(2), respectively. Gown, latex gloves, and UV-protective glasses showed dampening effects of 99.58%, 95.01%, and 99.73%, respectively. CXL appears to be a safe procedure with respect to UV-A radiation exposure to the surgeon. Further safety can be ensured by UV-protective devices.

Imaging and modeling of collagen architecture in living tissue with polarized light transfer (Conference Presentation)

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Ramella-Roman, Jessica C.; Stoff, Susan; Chue-Sang, Joseph; Bai, Yuqiang

2016-03-01

The extra-cellular space in connective tissue of animals and humans alike is comprised in large part of collagen. Monitoring of collagen arrangement and cross-linking has been utilized to diagnose a variety of medical conditions and guide surgical intervention. For example, collagen monitoring is useful in the assessment and treatment of cervical cancer, skin cancer, myocardial infarction, and non-arteritic anterior ischemic optic neuropathy. We have developed a suite of tools and models based on polarized light transfer for the assessment of collagen presence, cross-linking, and orientation in living tissue. Here we will present some example of such approach applied to the human cervix. We will illustrate a novel Mueller Matrix (MM) imaging system for the study of cervical tissue; furthermore we will show how our model of polarized light transfer through cervical tissue compares to the experimental findings. Finally we will show validation of the methodology through histological results and Second Harmonic imaging microscopy.

Treatment of chest burn contracture causing respiratory compromise with island release and grafting using cross-link collagen and Integra™ bilayer dressing

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Neil Doctor

2014-04-01

Full Text Available Post-burn skin contractures of the anterior and lateral abdomen and chest may result in respiratory compromise due to limitation of rib excursion. This case report describes a young man with respiratory compromise limiting his daily activity and function, as a result of a 90% burn sustained 6 years previously. Release of his chest and upper abdomen was achieved using “island” scar releases and a cross-linked bovine tendon collagen and glycosaminoglycan and a semi-permeable polysiloxane bilayer matrix dressing (Integra™ followed by subsequent split thickness skin graft. An immediate increase in maximal inspiratory volume was obtained intra-operatively and in the im­mediate post-operative period, and this improvement was sustained after healing of all wounds with subjective relief of the patient’s symptoms.

Current review and a simplified "five-point management algorithm" for keratoconus

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Rohit Shetty

2015-01-01

Full Text Available Keratoconus is a slowly progressive, noninflammatory ectatic corneal disease characterized by changes in corneal collagen structure and organization. Though the etiology remains unknown, novel techniques are continuously emerging for the diagnosis and management of the disease. Demographical parameters are known to affect the rate of progression of the disease. Common methods of vision correction for keratoconus range from spectacles and rigid gas-permeable contact lenses to other specialized lenses such as piggyback, Rose-K or Boston scleral lenses. Corneal collagen cross-linking is effective in stabilizing the progression of the disease. Intra-corneal ring segments can improve vision by flattening the cornea in patients with mild to moderate keratoconus. Topography-guided custom ablation treatment betters the quality of vision by correcting the refractive error and improving the contact lens fit. In advanced keratoconus with corneal scarring, lamellar or full thickness penetrating keratoplasty will be the treatment of choice. With such a wide spectrum of alternatives available, it is necessary to choose the best possible treatment option for each patient. Based on a brief review of the literature and our own studies we have designed a five-point management algorithm for the treatment of keratoconus.

Chitosan and thiolated chitosan: Novel therapeutic approach for preventing corneal haze after chemical injuries.

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Zahir-Jouzdani, Forouhe; Mahbod, Mirgholamreza; Soleimani, Masoud; Vakhshiteh, Faezeh; Arefian, Ehsan; Shahosseini, Saeed; Dinarvand, Rasoul; Atyabi, Fatemeh

2018-01-01

Corneal haze, commonly caused by deep physical and chemical injuries, can greatly impair vision. Growth factors facilitate fibroblast proliferation and differentiation, which leads to haze intensity. In this study, the potential effect of chitosan (CS) and thiolated-chitosan (TCS) nanoparticles and solutions on inhibition of fibroblast proliferation, fibroblast to myofibroblast differentiation, neovascularization, extracellular matrix (ECM) deposition, and pro-fibrotic cytokine expression was examined. Transforming growth factor beta-1 (TGFβ 1 ) was induced by interleukin-6 (IL6) in human corneal fibroblasts and expression levels of TGFβ 1 , Platelet-derived growth factor (PDGF), α-smooth muscle actins (α-SMA), collagen type I (Col I), fibronectin (Fn) and vascular endothelial growth factor (VEGF) were quantified using qRT-PCR. To assess wound-healing capacity, TCS-treated mice were examined for α-SMA positive cells, collagen deposition, inflammatory cells and neovascularization through pathological immunohistochemistry. The results revealed that CS and TCS could down-regulate the expression levels of TGFβ 1 and PDGF comparable to that of TGFβ 1 knockdown experiment. However, down-regulation of TGFβ 1 was not regulated through miR29b induction. Neovascularization along with α-SMA and ECM deposition were significantly diminished. According to these findings, CS and TCS can be considered as potential anti-fibrotic and anti-angiogenic therapeutics. Furthermore, TCS, thiolated derivative of CS, will increase mucoadhesion of the polymer at the corneal surface which makes the polymer efficient and non-toxic therapeutic approach for corneal injuries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Altered corneal biomechanical properties in children with osteogenesis imperfecta.

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Lagrou, Lisa M; Gilbert, Jesse; Hannibal, Mark; Caird, Michelle S; Thomas, Inas; Moroi, Sayoko E; Bohnsack, Brenda L

2018-04-07

To evaluate biomechanical corneal properties in children with osteogenesis imperfecta (OI). A prospective, observational, case-control study was conducted on children 6-19 years of age diagnosed with OI. Patients with OI and healthy control subjects underwent complete ophthalmic examinations. Additional tests included Ocular Response Analyzer (ORA) and ultrasonic pachymetry. Primary outcomes were central corneal thickness (CCT), corneal hysteresis (CH), and corneal resistance factor (CRF). Intraocular pressure (IOP) was measured directly by either iCare or Goldmann applanation and indirectly by the ORA (Goldmann-correlated and corneal-compensated IOP). Statistically significant differences between OI and control groups were determined using independent samples t test. A total of 10 of 18 OI cases (mean age, 13 ± 4.37 years; 8 males) and 30 controls (mean age, 12.76 ± 2.62 years; 16 males) were able to complete the corneal biomechanics and pachymetry testing. Children with OI had decreased CH (8.5 ± 1.0 mm Hg vs 11.6 ± 1.2 mm Hg [P < 0.001]), CRF (9.0 ± 1.9 mm Hg vs 11.5 ± 1.5 [P < 0.001]) and CCT (449.8 ± 30.8 μm vs 568 ± 47.6 μm [P < 0.001]) compared to controls. The corneal-compensated IOP was significantly higher in OI cases (18.8 ± 3.1 mm Hg) than in controls (15.0 ± 1.6 mm Hg, P < 0.004), but there was no significant difference in Goldmann-correlated IOP (16.3 ± 4.2 mm Hg vs 15.8 ± 2.2 mm Hg). Collagen defects in OI alter corneal structure and biomechanics. Children with OI have decreased CH, CRF, and CCT, resulting in IOPs that are likely higher than measured by tonometry. These corneal alterations are present at a young age in OI. Affected individuals should be routinely screened for glaucoma and corneal pathologies. Copyright © 2018 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering.

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Jui-Yang Lai

Full Text Available Hyaluronic acid (HA is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function.

Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates.

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Cavelier, S; Dastjerdi, A K; McKee, M D; Barthelat, F

2018-05-01

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive "binders" for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein - osteopontin (OPN) - appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates. Copyright © 2018 Elsevier Inc

In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins

International Nuclear Information System (INIS)

Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Soker, Shay; Khang, Gilson

2013-01-01

The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation. (paper)

Stability of Collagen Scaffold Implants for Animals with Iatrogenic Articular Cartilage Defects

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Josef Jančář

2009-01-01

Full Text Available Synthesis and characterization of biodegradable hydrogels based on collagen modified by addition of synthetic biodegradable copolymer intended for preparation of porous scaffolds for mesenchymal stem cells used for possible implantation to animals with articular surface defects was investigated. The synthetic biodegradable tri-block copolymer used was the block copolymer of polyethylene glycol (PEG, polylactic acid (PLA, polyglycolic acid (PGA (PEG-PLGA endcapped with itaconic acid (ITA. The water-soluble carbodiimide and N-hydroxysuccimide system (EDC-NHS was chosen as the cross-linking agent used to control the rate of hydrogel resorption. Dependence of the physical properties of the prepared hydrogels on the concentration of the EDC-NHS cross-linker, reaction time and concentration of PEG-PLGA-ITA copolymer was examined. Swelling behaviour, thermal stability, surface morphology and degradation rate were also characterized. Based on the obtained results, it can be concluded that increase in concentration of the cross-linking agent, as well as prolonged cross-linking time and increased amount of synthetic copolymer lead to enhanced thermal stability of the gels together with a reduced swelling ratio and degradation rate in saline. The resorption rate of these gels used in preparation of cartilage scaffolds can be controlled over a wide time interval by varying the collagen/(PEG-PLGA-ITA blend composition or the conditions of the cross-linking reaction.

Chitosan-Coated Collagen Membranes Promote Chondrocyte Adhesion, Growth, and Interleukin-6 Secretion

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Nabila Mighri

2015-11-01

Full Text Available Designing scaffolds made from natural polymers may be highly attractive for tissue engineering strategies. We sought to produce and characterize chitosan-coated collagen membranes and to assess their efficacy in promoting chondrocyte adhesion, growth, and cytokine secretion. Porous collagen membranes were placed in chitosan solutions then crosslinked with glutaraldehyde vapor. Fourier transform infrared (FTIR analyses showed elevated absorption at 1655 cm-1 of the carbon–nitrogen (N=C bonds formed by the reaction between the (NH2 of the chitosan and the (C=O of the glutaraldehyde. A significant peak in the amide II region revealed a significant deacetylation of the chitosan. Scanning electron microscopy (SEM images of the chitosan-coated membranes exhibited surface variations, with pore size ranging from 20 to 50 µm. X-ray photoelectron spectroscopy (XPS revealed a decreased C–C groups and an increased C–N/C–O groups due to the reaction between the carbon from the collagen and the NH2 from the chitosan. Increased rigidity of these membranes was also observed when comparing the chitosan-coated and uncoated membranes at dried conditions. However, under wet conditions, the chitosan coated collagen membranes showed lower rigidity as compared to dried conditions. Of great interest, the glutaraldehyde-crosslinked chitosan-coated collagen membranes promoted chondrocyte adhesion, growth, and interleukin (IL-6 secretion. Overall results confirm the feasibility of using designed chitosan-coated collagen membranes in future applications, such as cartilage repair.

Production and in vitro characterization of 3D porous scaffolds made of magnesium carbonate apatite (MCA)/anionic collagen using a biomimetic approach

International Nuclear Information System (INIS)

Sader, Marcia S.; Martins, Virginia C.A.; Gomez, Santiago; LeGeros, Racquel Z.; Soares, Gloria A.

2013-01-01

3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ∼ 10 nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity. Highlights: • 3D scaffolds of Mg-carbonate–apatite and anionic-collagen were produced. • The biomimetic approach and the cross-linking with 0.25% GA solution were employed. • The scaffolds showed open-porous structure and apatite crystals on collagen fibrils. • The cross-linked scaffolds exhibited integrity when immersed in culture medium. • SaOs2 cells adhered and proliferated on the cross-linked scaffolds confirming no cytotoxicity

Production and in vitro characterization of 3D porous scaffolds made of magnesium carbonate apatite (MCA)/anionic collagen using a biomimetic approach

Energy Technology Data Exchange (ETDEWEB)

Sader, Marcia S., E-mail: msader@metalmat.ufrj.br [Prog. Engenharia Metalúrgica e Materiais, COPPE/UFRJ, RJ (Brazil); Martins, Virginia C.A. [Depto. de Química e Física Molecular, IQSC/USP, SP (Brazil); Gomez, Santiago [Dept. Anatomía Patológica, Universidad de Cádiz, Cadiz (Spain); LeGeros, Racquel Z. [Department of Biomaterials and Biomimetics, New York University College of Dentistry, NY (United States); Soares, Gloria A. [Prog. Engenharia Metalúrgica e Materiais, COPPE/UFRJ, RJ (Brazil)

2013-10-15

3D porous scaffolds are relevant biomaterials to bone engineering as they can be used as templates to tissue reconstruction. The aim of the present study was to produce and characterize in vitro 3D magnesium-carbonate apatite/collagen (MCA/col) scaffolds. They were prepared by using biomimetic approach, followed by cross-linking with 0.25% glutaraldehyde solution (GA) and liofilization. Results obtained with Fourier-transform infrared spectroscopy (FT-IR) confirmed the type-B carbonate substitution, while by X-ray diffraction (XRD), a crystallite size of ∼ 10 nm was obtained. Optical and electron microscopy showed that the cylindrical samples exhibited an open-porous morphology, with apatite nanocrystals precipitated on collagen fibrils. The cross-linked 3D scaffolds showed integrity when immersed in culture medium up to 14 days. Also, the immersion of such samples into an acid buffer solution, to mimic the osteoclastic resorption environment, promotes the release of important ions for bone repair, such as calcium, phosphorus and magnesium. Bone cells (SaOs2) adhered, and proliferated on the 3D composite scaffolds, showing that synthesis and the cross-linking processes did not induce cytotoxicity. Highlights: • 3D scaffolds of Mg-carbonate–apatite and anionic-collagen were produced. • The biomimetic approach and the cross-linking with 0.25% GA solution were employed. • The scaffolds showed open-porous structure and apatite crystals on collagen fibrils. • The cross-linked scaffolds exhibited integrity when immersed in culture medium. • SaOs2 cells adhered and proliferated on the cross-linked scaffolds confirming no cytotoxicity.

Enhanced Biological Response of AVS-Functionalized Ti-6Al-4V Alloy through Covalent Immobilization of Collagen.

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Rezvanian, Parsa; Daza, Rafael; López, Patricia A; Ramos, Milagros; González-Nieto, Daniel; Elices, Manuel; Guinea, Gustavo V; Pérez-Rigueiro, José

2018-02-20

This study presents the development of an efficient procedure for covalently immobilizing collagen molecules on AVS-functionalized Ti-6Al-4V samples, and the assessment of the survival and proliferation of cells cultured on these substrates. Activated Vapor Silanization (AVS) is a versatile functionalization technique that allows obtaining a high density of active amine groups on the surface. A procedure is presented to covalently bind collagen to the functional layer using EDC/NHS as cross-linker. The covalently bound collagen proteins are characterized by fluorescence microscopy and atomic force microscopy and their stability is tested. The effect of the cross-linker concentration on the process is assessed. The concentration of the cross-linker is optimized and a reliable cleaning protocol is developed for the removal of the excess of carbodiimide from the samples. The results demonstrate that the covalent immobilization of collagen type I on Ti-6Al-4V substrates, using the optimized protocol, increases the number of viable cells present on the material. Consequently, AVS in combination with the carbodiimide chemistry appears as a robust method for the immobilization of proteins and, for the first time, it is shown that it can be used to enhance the biological response to the material.

Bilateral congenital corneal keloids and anterior segment mesenchymal dysgenesis in a case of Rubinstein-Taybi syndrome.

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Rao, Srinivas K; Fan, Dorothy S P; Pang, C P; Li, Winnie W Y; Ng, Joan S K; Good, William V; Lam, Dennis S C

2002-01-01

To report the unusual association of bilateral corneal keloids and anterior segment mesenchymal dysgenesis in a child with Rubinstein-Taybi syndrome. Case report of a 2-year-old boy. Excision of the epicorneal mass in the right eye was followed by recurrence of the lesion. Multiple penetrating keratoplasties were unsuccessful in reconstructing the anterior segment because of recurrent corneal epithelial breakdown, suggesting limbal stem cell insufficiency. Histopathology and electron microscopy of the excised mass lesion showed features typical of a corneal keloid: thickened keratinized epithelium, absent Bowman's layer, and fibrovascular hyperplasia, with haphazard orientation of the collagen lamellae. Ultrasound biomicroscopy and intraoperative findings suggested a diagnosis of Peter anomaly, but genetic analysis did not show a PAX6 mutation. The findings in our patient add to the spectrum of ocular changes described in Rubinstein-Taybi syndrome and confirm earlier reports of poor ocular prognosis in corneal keloids and Rubinstein-Taybi syndrome.

Removal of the basement membrane enhances corneal wound healing.

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Pal-Ghosh, Sonali; Pajoohesh-Ganji, Ahdeah; Tadvalkar, Gauri; Stepp, Mary Ann

2011-12-01

Recurrent corneal erosions are painful and put patients' vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, β4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely. Copyright © 2011 Elsevier Ltd. All rights reserved.

Response of human corneal fibroblasts on silk film surface patterns.

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Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L

2010-06-11

Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.

New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS.

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Kaczmarek, B; Sionkowska, A; Kozlowska, J; Osyczka, A M

2018-02-01

Nowadays, fabrication of composite materials based on biopolymers is a rising field due to potential for bone repair and tissue engineering application. Blending of different biopolymers and incorporation of inorganic particles in the blend can lead to new materials with improved physicochemical properties and biocompatibility. In this work 3D porous structures called scaffolds based on chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Scaffolds were cross-linked by EDC/NHS. Infrared spectra for the materials were made, the percentage of swelling, scaffolds porosity and density, mechanical parameters, thermal stability were studied. Moreover, the scaffolds were used as matrixes for the calcium phosphate in situ precipitation. SEM images were taken and EDX analysis was carried out for calcium and phosphorous content determination in the scaffold. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on obtained scaffolds. The results showed that the properties of 3D composites cross-linked by EDC/NHS were altered after the addition of 1, 2 and 5% hyaluronic acid. Mechanical parameters, thermal stability and porosity of scaffolds were improved. Moreover, calcium and phosphorous were found in each kind of scaffold. SEM images showed that the precipitation was homogeneously carried in the whole volume of samples. Attachment of SaOS-2 cells to all modified materials was better compared to unmodified control and proliferation of these cells was markedly increased on scaffolds with precipitated calcium phosphate. Obtained materials can provide the support useful in tissue engineering and regenerative medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of human collagen biomaterials in the healing of colonic anastomoses in dogs.

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Mutter, D; Aprahamian, M; Tiollier, J; Sonzini, P; Marescaux, J

1997-04-01

To investigate the ability of human collagen biomaterials to secure colonic anastomoses in dogs and to evaluate the biocompatibility of anastomotic protection patches (APP). Experimental open study. Experimental research centre, France. 21 mongrel dogs randomised into three groups of 7 each. Standard transverse colonic end-to-end anastomoses were secured with two-layer oxidised collagen I + III sponge covered with thin crosslinked collagen IV film (APP 1) glued around the suture (n = 7); two-layer oxidised collagen I + III sponge covered with thin non-crosslinked collagen I + III film patch (APP 2) (n = 7); or sealed by fibrin sealant (n = 7), which acted as a controls. Gross examination, radiological control (barium enemas), and microscopic examination on day 35 postoperatively. Gross clinical and radiological examinations on day 35 showed normal wound healing in all but one dog in which the anastomoses had occluded by day 16. There was significantly less stricturing with the APP 2 patch (p < 0.05 compared with the controls). Microscopic examination showed complete absorption of the APP 2 patches as well as quicker mucosal and extracellular matrix repair than controls. The APP 1 patch gave the best healing of the muscular layer but did not reduce anastomosis stricturing, and was not totally absorbed. Collagen supporting devices do not alter healing of the large bowel. Encircling patches do not increase the number of adhesions or the rate of anastomotic stricturing and a thin fibrillar collagen I + III dense layer may even improve it. The speed of absorption of the patch depends on the type of dense collagen film. These results argue for a prospective clinical evaluation in humans.

Serum cross-linked n-telopeptides of type 1 collagen (NTx in patients with solid tumors

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Fernando Jablonka

Full Text Available CONTEXT AND OBJECTIVE: Cross-linked N-telopeptides of type I collagen (NTx increase in concentration in situations in which bone resorption is increased, such as osteoporosis and bone metastasis (BM. We aimed to evaluate the serum concentrations of NTx in a sample of patients with several types of solid tumors. DESIGN AND SETTING: Cross-sectional analytical study with a control group in a tertiary public hospital. METHODS: We performed the quantitative enzyme-linked immunosorbent assay (ELISA on serum NTx levels in 19 subjects without a history of cancer and 62 patients with various solid tumors who had been referred for a bone scan. Three experienced analysts read all bone scans. RESULTS: The serum NTx levels in patients with cancer and BM, with cancer but without BM and without cancer were 46.77 ± 2.58, 32.85 ± 2.05 and 22.32 ± 2.90 respectively (P < 0.0001. We did not find any significant correlations of serum NTx with age, gender, history of bone pain, tumor type and bone alkaline phosphatase levels. We found a significant correlation between serum NTx and alkaline phosphatase levels (R² = 0.08; P = 0.022. CONCLUSIONS: Serum NTx levels are significantly higher in patients with solid tumors and bone metastases than they are in patients without bone metastases and in normal controls.

A Simple and Efficient Method to Improve Mechanical Properties of Collagen Scaffolds by UV Irradiation

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F. Khayyatan

2010-12-01

Full Text Available Collagen is the major protein component of cartilage, bone, skin and connective tissue and constitutes the major part of the extracellular matrix. Collagen type I has complex structural hierarchy, which consists of treepolypeptide α-chains wound together in a rod-like helical structure. Collagen is an important biomaterial, finding many applications in the field of tissue engineering. It has been processed into various shapes, such as, gel, film, sponge and fiber. It is commonly used as the scaffolding material for tissue engineering due to its many superior properties including low antigenicity and high growth promotion. Unfortunately, poor mechanical properties and rapid degradation rates of collagen scaffolds can cause instability and difficulty in handling. By crosslinking, the structural stability of the collagen and its rate of resorption can be adapted with respect to its demanding requirements. The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. In thisstudy, the effect of UV irradiation on collagen scaffolds has been carried out.Collagen scaffolds were fabricated using freeze drying method with freezing temperature of -80oC, then exposed to UV irradiation. Mean pore size of the scaffolds was obtained as 98.52±14.51 μm using scanning electron microscopy. Collagen scaffolds exposed to UV Irradiation (254 nm for 15 min showed the highest tensile strain (17.37±0.98 %, modulus (1.67±0.15 MPa and maximum load (24.47±2.38 cN values. As partial loss of the native collagen structure may influence attachment, migration, and proliferation of cells on collagen scaffolds, we detected no intact α-chains after SDS-Page chromatography. We demonstrate that UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen scaffolds without any molecular fracture.

Collagen Fibrils: Nature's Highly Tunable Nonlinear Springs.

Science.gov (United States)

Andriotis, Orestis G; Desissaire, Sylvia; Thurner, Philipp J

2018-03-21

Tissue hydration is well known to influence tissue mechanics and can be tuned via osmotic pressure. Collagen fibrils are nature's nanoscale building blocks to achieve biomechanical function in a broad range of biological tissues and across many species. Intrafibrillar covalent cross-links have long been thought to play a pivotal role in collagen fibril elasticity, but predominantly at large, far from physiological, strains. Performing nanotensile experiments of collagen fibrils at varying hydration levels by adjusting osmotic pressure in situ during atomic force microscopy experiments, we show the power the intrafibrillar noncovalent interactions have for defining collagen fibril tensile elasticity at low fibril strains. Nanomechanical tensile tests reveal that osmotic pressure increases collagen fibril stiffness up to 24-fold in transverse (nanoindentation) and up to 6-fold in the longitudinal direction (tension), compared to physiological saline in a reversible fashion. We attribute the stiffening to the density and strength of weak intermolecular forces tuned by hydration and hence collagen packing density. This reversible mechanism may be employed by cells to alter their mechanical microenvironment in a reversible manner. The mechanism could also be translated to tissue engineering approaches for customizing scaffold mechanics in spatially resolved fashion, and it may help explain local mechanical changes during development of diseases and inflammation.

Immune responses to implanted human collagen graft in rats

International Nuclear Information System (INIS)

Quteish, D.; Dolby, A.E.

1991-01-01

Immunity to collagen implants may be mediated by cellular and humoral immune responses. To examine the possibility of such immunological reactivity and crossreactivity to collagen, 39 Sprague-Dawley rats (female, 10 weeks old, approximately 250 g wt) were implanted subcutaneously at thigh sites with crosslinked, freeze-dried human placental type I collagen grafts (4x4x2 mm) which had been irradiated (520 Gray) or left untreated. Blood was obtained by intracardiac sampling prior to implantation or from normal rats, and at various times afterwards when the animals were sacrificed. The sera from these animals were examined for circulating antibodies to human, bovine and rat tail (type I) collagens by enzyme-linked immunosorbent assay (ELISA). Also, the lymphoblastogenic responses of spleen lymphocytes from the irradiated collagen-implanted animals were assessed in culture by measuring thymidine uptake with autologous and normal rat sera in the presence of human bovine type I collagens. Implantation of the irradiated and non-irradiated collagen graft in rats led to a significant increase in the level of circulating antibodies to human collagen. Also antibody to bovine and rat tail collagens was detectable in the animals implanted with irradiated collagen grafts but at a lower level than the human collagen. There was a raised lymphoblastogenic response to both human and bovine collagens. The antibody level and lymphoblastogenesis to the tested collagens gradually decreased towards the end of the post-implantation period. (author)

Collagen-Gold Nanoparticle Conjugates for Versatile Biosensing

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Sarah Unser

2017-02-01

Full Text Available Integration of noble metal nanoparticles with proteins offers promising potential to create a wide variety of biosensors that possess both improved selectivity and versatility. The multitude of functionalities that proteins offer coupled with the unique optical properties of noble metal nanoparticles can allow for the realization of simple, colorimetric sensors for a significantly larger range of targets. Herein, we integrate the structural protein collagen with 10 nm gold nanoparticles to develop a protein-nanoparticle conjugate which possess the functionality of the protein with the desired colorimetric properties of the nanoparticles. Applying the many interactions that collagen undergoes in the extracellular matrix, we are able to selectively detect both glucose and heparin with the same collagen-nanoparticle conjugate. Glucose is directly detected through the cross-linking of the collagen fibrils, which brings the attached nanoparticles into closer proximity, leading to a red-shift in the LSPR frequency. Conversely, heparin is detected through a competition assay in which heparin-gold nanoparticles are added to solution and compete with heparin in the solution for the binding sites on the collagen fibrils. The collagen-nanoparticle conjugates are shown to detect both glucose and heparin in the physiological range. Lastly, glucose is selectively detected in 50% mouse serum with the collagen-nanoparticle devices possessing a linear range of 3–25 mM, which is also within the physiologically relevant range.

Influence of tetrahydrocurcumin on tail tendon collagen contents and its properties in rats with streptozotocin-nicotinamide-induced type 2 diabetes.

Science.gov (United States)

Pari, Leelavinothan; Murugan, Pidaran

2007-12-01

Changes in the structural and functional properties of collagen caused by advanced glycation might be of importance for the etiology of late-stage complications in diabetics. Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, exhibiting many of the same physiological and pharmacological activities of curcumin and in some systems may exert greater antioxidant activity than curcumin. In diabetic rats, hydroxyproline and collagen content as well as its degree of cross-linking were increased, as shown by increased extent of glycation, collagen-linked fluorescence, neutral salt collagen, and decreased acid and pepsin solubility. Administration of THC for 45 days to diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effects of THC were comparable with those of curcumin. In conclusion, administration of THC had a positive influence on the content of collagen and its properties in streptozotocin- and nicotinamide-induced diabetic rats. THC was found to be more effective than curcumin.

Redundancy and cooperativity in the mechanics of compositely crosslinked filamentous networks.

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Moumita Das

Full Text Available The cytoskeleton of living cells contains many types of crosslinkers. Some crosslinkers allow energy-free rotations between filaments and others do not. The mechanical interplay between these different crosslinkers is an open issue in cytoskeletal mechanics. Therefore, we develop a theoretical framework based on rigidity percolation to study a generic filamentous system containing both stretching and bond-bending forces to address this issue. The framework involves both analytical calculations via effective medium theory and numerical simulations on a percolating triangular lattice with very good agreement between both. We find that the introduction of angle-constraining crosslinkers to a semiflexible filamentous network with freely rotating crosslinks can cooperatively lower the onset of rigidity to the connectivity percolation threshold-a result argued for years but never before obtained via effective medium theory. This allows the system to ultimately attain rigidity at the lowest concentration of material possible. We further demonstrate that introducing angle-constraining crosslinks results in mechanical behaviour similar to just freely rotating crosslinked semflexible filaments, indicating redundancy and universality. Our results also impact upon collagen and fibrin networks in biological and bio-engineered tissues.

Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

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Hongshan Liu

Full Text Available BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Corneal Laceration

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Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone

Energy Technology Data Exchange (ETDEWEB)

Willems, Nop M.B.K., E-mail: n.willems@acta.nl [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Langenbach, Geerling E.J. [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Stoop, Reinout [Dept. of Metabolic Health Research, TNO, P.O. Box 2215, 2301 CE Leiden (Netherlands); Toonder, Jaap M.J. den [Dept. of Mechanical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Mulder, Lars [Dept. of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Zentner, Andrej [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Everts, Vincent [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands)

2014-09-01

The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2 M ribose at 37 °C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness. - Highlights: • The assessment of effects of glycation in bone using HPLC, microCT, and nanoindentation • Ribose incubation: 300‐fold increase in the number of pentosidine cross-links • 300‐fold increase in the number of pentosidine cross-links: no changes in bone tissue stiffness.

Biophysical behavior of Scomberoides commersonianus skin collagen.

Science.gov (United States)

Kolli, Nagamalleswari; Joseph, K Thomas; Ramasami, T

2002-06-01

Some biophysical characteristics of the skin collagen from Scomberoides commersonianus were measured and compared to those of rat tail tendon. Stress-strain data indicate that the strain at break as well as the tensile strength of the fish skin without scales increased significantly. The maximum tension in case of rat skin is at least a factor of two higher than that observed in fish skin. The much lower hydrothermal isometric tension measurements observed in fish skin are attributable to a lesser number of heat stable crosslinks. Stress relaxation measurements in the fish skin indicate that more than one relaxation process may be involved in the stabilization of collagenous matrix. The observed differences in the biophysical behavior of fish skin may well arise from combination of changes in extent of hydroxylation of proline in collagen synthesis, hydrogen bond network and fibril orientation as compared to rat tail tendon.

The effect of irradiation on rheological and electrical properties of collagen

Czech Academy of Sciences Publication Activity Database

Landfeld, A.; Houska, M.; Skočilas, J.; Žitný, R.; Novotná, P.; Stangl, J.; Dostál, M.; Chvátil, David

2016-01-01

Roč. 26, č. 4 (2016), s. 43775 ISSN 1430-6395 Institutional support: RVO:61389005 Keywords : Bovine collagen * electrical conductivity * storage modulus * loss modulus * electron irradiation * crosslinking Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 1.222, year: 2016

N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

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Bernadette K. Madathil

2014-01-01

Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

Spontaneous Corneal Hydrops in a Patient with a Corneal Ulcer

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Hatim Batawi

2016-01-01

Full Text Available Purpose: We report the case of a 77-year-old man with no history of keratoconus or other ectatic disorders who presented with corneal hydrops in the setting of a corneal ulcer. The risk factors, pathogenesis and treatment options of corneal hydrops are discussed. Method: This is an observational case report study. Results: A 77-year-old man presented with a 1-day history of severe pain, redness, mucous discharge and photophobia in the right eye. A slit-lamp examination of the right eye showed an area of focal corneal edema and protrusion. Within the area of edema and protrusion, there was an infiltrate with an overlying epithelial defect consistent with an infectious corneal ulcer. The Seidel test showed no leakage, so a clinical diagnosis of corneal hydrops associated with nonperforated corneal ulcer was made. With appropriate antibiotic treatment, the corneal ulcer and hydrops both resolved over a 1-month period. Conclusion: Corneal hydrops can occur in the setting of corneal infections.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

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André Alves Dias

2012-12-01

Full Text Available When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp, a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Embryonic chicken cornea and cartilage synthesize type IX collagen molecules with different amino-terminal domains.

OpenAIRE

Svoboda, K K; Nishimura, I; Sugrue, S P; Ninomiya, Y; Olsen, B R

1988-01-01

We have analyzed embryonic chicken cornea for the presence of type IX collagen mRNA and protein. Using RNA transfer blot analysis, we demonstrate that alpha 1(IX) and alpha 2(IX) mRNAs are expressed by corneal epithelial cells at the time that the primary stromal components are synthesized. The levels of the mRNAs decrease with increasing developmental age and are barely detectable at day 11 of development. In contrast, type IX collagen protein is detectable by immunofluorescence at days 5 an...

Corneal Laceration

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Shape-Memory Effects in Biopolymer Networks with Collagen-Like Transient Nodes

NARCIS (Netherlands)

Skrzeszewska, P.J.; Jong, L.N.; Wolf, de F.A.; Stuart, M.A.C.; Gucht, van der J.

2011-01-01

In this article we study shape-memory behavior of hydrogels, formed by biodegradable and biocompatible recombinant telechelic polypeptides, with collagen-like end blocks and a random coil-like middle block. The programmed shape of these hydrogels was achieved by chemical cross-linking of lysine

Effect of Vaginal or Systemic Estrogen on Dynamics of Collagen Assembly in the Rat Vaginal Wall1

Science.gov (United States)

Montoya, T. Ignacio; Maldonado, P. Antonio; Acevedo, Jesus F.; Word, R. Ann

2014-01-01

ABSTRACT The objective of this study was to compare the effects of systemic and local estrogen treatment on collagen assembly and biomechanical properties of the vaginal wall. Ovariectomized nulliparous rats were treated with estradiol or conjugated equine estrogens (CEEs) either systemically, vaginal CEE, or vaginal placebo cream for 4 wk. Low-dose local CEE treatment resulted in increased vaginal epithelial thickness and significant vaginal growth without uterine hyperplasia. Furthermore, vaginal wall distensibility increased without compromise of maximal force at failure. Systemic estradiol resulted in modest increases in collagen type I with no change in collagen type III mRNA. Low-dose vaginal treatment, however, resulted in dramatic increases in both collagen subtypes whereas moderate and high dose local therapies were less effective. Consistent with the mRNA results, low-dose vaginal estrogen resulted in increased total and cross-linked collagen content. The inverse relationship between vaginal dose and collagen expression may be explained in part by progressive downregulation of estrogen receptor-alpha mRNA with increasing estrogen dose. We conclude that, in this menopausal rat model, local estrogen treatment increased total and cross-linked collagen content and markedly stimulated collagen mRNA expression in an inverse dose-effect relationship. High-dose vaginal estrogen resulted in downregulation of estrogen receptor-alpha and loss of estrogen-induced increases in vaginal collagen. These results may have important clinical implications regarding the use of local vaginal estrogen therapy and its role as an adjunctive treatment in women with loss of vaginal support. PMID:25537371

Evaluation of proanthocyanidin-crosslinked electrospun gelatin nanofibers for drug delivering system

International Nuclear Information System (INIS)

Huang, Chiung-Hua; Chi, Chin-Ying; Chen, Yueh-Sheng; Chen, Kuo-Yu; Chen, Pei-Lain; Yao, Chun-Hsu

2012-01-01

Electrospun nanofibers are excellent candidates for various biomedical applications. We successfully fabricated proanthocyanidin‐crosslinked gelatin electrospun nanofibers. Proanthocyanidin, a low cytotoxic collagen crosslinking reagent, increased the gelatin crosslinking percentage in the nanofibers from 53% to 64%. The addition of proanthocyanidin kept the nanofibers from swelling, and, thus, made the fibers more stable in the aqueous state. The compatibility and the release behavior of the drug in the nanofibers were examined using magnesium ascorbyl phosphate as the model drug. Proanthocyanidin also promoted drug loading and kept the drug release rate constant. These properties make the proanthocyanidin‐crosslinked gelatin nanofibers an excellent material for drug delivery. In the cell culture study, L929 fibroblast cells had a significantly higher proliferation rate when cultured with the gelatin/proanthocyanidin blended nanofibers. This characteristic showed that proanthocyanidin‐crosslinked gelatin electrospun nanofibers could potentially be employed as a wound healing material by increasing cell spreading and proliferation. - Highlights: ► Proanthocyanidin‐crosslinked gelatin nanofibers (GEL/PA) is synthesized. ► Proanthocyanidin promoted drug loading and kept the drug release rate constant. ► The GEL/PA nanofibers accelerate fibroblast cell proliferation. ► The GEL/PA nanofibers increase the drug loading efficiency.

Nanorod mediated collagen scaffolds as extra cellular matrix mimics

International Nuclear Information System (INIS)

Vedhanayagam, Mohan; Nair, Balachandran Unni; Sreeram, Kalarical Janardhanan; Mohan, Ranganathan

2015-01-01

Creating collagen scaffolds that mimic extracellular matrices without using toxic exogenous materials remains a big challenge. A new strategy to create scaffolds through end-to-end crosslinking through functionalized nanorods leading to well-designed architecture is presented here. Self-assembled scaffolds with a denaturation temperature of 110 °C, porosity of 70%, pore size of 0.32 μm and Young’s modulus of 231 MPa were developed largely driven by imine bonding between 3-mercapto-1-propanal (MPA) functionalized ZnO nanorods and collagen. The mechanical properties obtained were much higher than that of native collagen, collagen—MPA, collagen—3-mercapto-1-propanol (3MPOH) or collagen- 3-MPOH-ZnO, clearly bringing out the relevance of nanorod mediated assembly of fibrous networks. This new strategy has led to scaffolds with mechanical properties much higher than earlier reports and can provide support for cell growth and facilitation of cell attachment. (paper)

ZEB1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis.

Science.gov (United States)

Peng, D H; Ungewiss, C; Tong, P; Byers, L A; Wang, J; Canales, J R; Villalobos, P A; Uraoka, N; Mino, B; Behrens, C; Wistuba, I I; Han, R I; Wanna, C A; Fahrenholtz, M; Grande-Allen, K J; Creighton, C J; Gibbons, D L

2017-04-06

Lung cancer is the leading cause of cancer-related deaths, primarily due to distant metastatic disease. Metastatic lung cancer cells can undergo an epithelial-to-mesenchymal transition (EMT) regulated by various transcription factors, including a double-negative feedback loop between the microRNA-200 (miR-200) family and ZEB1, but the precise mechanisms by which ZEB1-dependent EMT promotes malignancy remain largely undefined. Although the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. Investigating the collaborative effect of EMT and ECM in the metastatic process reveals increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in ZEB1-activated mesenchymal lung cancer cells. In addition, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and ZEB1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principal isoform that crosslinks and stabilizes insoluble collagen deposition in tumor tissues. In turn, focal adhesion formation and FAK/SRC signaling is activated in mesenchymal tumor cells by crosslinked collagen in the ECM. Our study is the first to validate direct regulation of LOX and LOXL2 by the miR-200/ZEB1 axis, defines a novel mechanism driving tumor metastasis, delineates collagen as a prognostic marker, and identifies LOXL2 as a potential therapeutic target against tumor progression.

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces.

Science.gov (United States)

Ban, Ehsan; Franklin, J Matthew; Nam, Sungmin; Smith, Lucas R; Wang, Hailong; Wells, Rebecca G; Chaudhuri, Ovijit; Liphardt, Jan T; Shenoy, Vivek B

2018-01-23

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mechanisms of lamellar collagen formation in connective tissues.

Science.gov (United States)

Ghazanfari, Samaneh; Khademhosseini, Ali; Smit, Theodoor H

2016-08-01

The objective of tissue engineering is to regenerate functional tissues. Engineering functional tissues requires an understanding of the mechanisms that guide the formation and evolution of structure in the extracellular matrix (ECM). In particular, the three-dimensional (3D) collagen fiber arrangement is important as it is the key structural determinant that provides mechanical integrity and biological function. In this review, we survey the current knowledge on collagen organization mechanisms that can be applied to create well-structured functional lamellar tissues and in particular intervertebral disc and cornea. Thus far, the mechanisms behind the formation of cross-aligned collagen fibers in the lamellar structures is not fully understood. We start with cell-induced collagen alignment and strain-stabilization behavior mechanisms which can explain a single anisotropically aligned collagen fiber layer. These mechanisms may explain why there is anisotropy in a single layer in the first place. However, they cannot explain why a consecutive collagen layer is laid down with an alternating alignment. Therefore, we explored another mechanism, called liquid crystal phasing. While dense concentrations of collagen show such behavior, there is little evidence that the conditions for liquid crystal phasing are actually met in vivo. Instead, lysyl aldehyde-derived collagen cross-links have been found essential for correct lamellar matrix deposition. Furthermore, we suggest that supra-cellular (tissue-level) shear stress may be instrumental in the alignment of collagen fibers. Understanding the potential mechanisms behind the lamellar collagen structure in connective tissues will lead to further improvement of the regeneration strategies of functional complex lamellar tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

[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

D-Glucose as a modifying agent in gelatin/collagen matrix and reservoir nanoparticles for Calendula officinalis delivery.

Science.gov (United States)

Lam, P-L; Kok, S H-L; Bian, Z-X; Lam, K-H; Tang, J C-O; Lee, K K-H; Gambari, R; Chui, C-H

2014-05-01

Gelatin/Collagen-based matrix and reservoir nanoparticles require crosslinkers to stabilize the formed nanosuspensions, considering that physical instability is the main challenge of nanoparticulate systems. The use of crosslinkers improves the physical integrity of nanoformulations under the-host environment. Aldehyde-based fixatives, such as formaldehyde and glutaraldehyde, have been widely applied to the crosslinking process of polymeric nanoparticles. However, their potential toxicity towards human beings has been demonstrated in many previous studies. In order to tackle this problem, D-glucose was used during nanoparticle formation to stabilize the gelatin/collagen-based matrix wall and reservoir wall for the deliveries of Calendula officinalis powder and oil, respectively. In addition, therapeutic selectivity between malignant and normal cells could be observed. The C. officinalis powder loaded nanoparticles significantly strengthened the anti-cancer effect towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells when compared with the free powder. On the contrary, the nanoparticles did not show significant cytotoxicity towards normal esophageal epithelial NE3 cells and human skin keratinocyte HaCaT cells. On the basis of these evidences, D-glucose modified gelatin/collagen matrix nanoparticles containing C. officinalis powder might be proposed as a safer alternative vehicle for anti-cancer treatments. Copyright © 2014 Elsevier B.V. All rights reserved.

A three-dimensional collagen-fiber network model of the extracellular matrix for the simulation of the mechanical behaviors and micro structures.

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Dong, Shoubin; Huang, Zetao; Tang, Liqun; Zhang, Xiaoyang; Zhang, Yongrou; Jiang, Yi

2017-07-01

The extracellular matrix (ECM) provides structural and biochemical support to cells and tissues, which is a critical factor for modulating cell dynamic behavior and intercellular communication. In order to further understand the mechanisms of the interactive relationship between cell and the ECM, we developed a three-dimensional (3D) collagen-fiber network model to simulate the micro structure and mechanical behaviors of the ECM and studied the stress-strain relationship as well as the deformation of the ECM under tension. In the model, the collagen-fiber network consists of abundant random distributed collagen fibers and some crosslinks, in which each fiber is modeled as an elastic beam and a crosslink is modeled as a linear spring with tensile limit, it means crosslinks will fail while the tensile forces exceed the limit of spring. With the given parameters of the beam and the spring, the simulated tensile stress-strain relation of the ECM highly matches the experimental results including damaged and failed behaviors. Moreover, by applying the maximal inscribed sphere method, we measured the size distribution of pores in the fiber network and learned the variation of the distribution with deformation. We also defined the alignment of the collagen-fibers to depict the orientation of fibers in the ECM quantitatively. By the study of changes of the alignment and the damaged crosslinks against the tensile strain, this paper reveals the comprehensive mechanisms of four stages of 'toe', 'linear', 'damage' and 'failure' in the tensile stress-strain relation of the ECM which can provide further insight in the study of cell-ECM interaction.

Betanin reduces the accumulation and cross-links of collagen in high-fructose-fed rat heart through inhibiting non-enzymatic glycation.

Science.gov (United States)

Han, Junyan; Tan, Chang; Wang, Yiheng; Yang, Shaobin; Tan, Dehong

2015-02-05

We attempted to determine whether betanin (from natural pigments) that has antioxidant properties would be protective against fructose-induced diabetic cardiac fibrosis in Sprague-Dawley rats. Fructose water solution (30%) was accessed freely, and betanin (25 and 100 mg/kg/d) was administered by intra-gastric gavage continuously for 60 d. Rats were sacrificed after overnight fast. The rat blood and left ventricle were collected. In vitro antiglycation assay in bovine serum albumin/fructose system was also performed. In rats treated only with fructose, levels of plasma markers: glucose, insulin, HOMA and glycated hemoglobin rised, left ventricle collagen accumulated and cross-linked, profibrotic factor-transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) protein expression increased, and soluble collagen decreased, compared with those in normal rats, showing fructose induces diabetic cardiac fibrosis. Treatment with betanin antagonized the changes of these parameters, demonstrating the antifibrotic role of betanin in the selected diabetic models. In further mechanistic study, betanin decreased protein glycation indicated by the decreased levels of protein glycation reactive intermediate (methylglyoxal), advanced glycation end product (N(ε)-(carboxymethyl) lysine) and receptors for advanced glycation end products (AGEs), antagonized oxidative stress and nuclear factor-κB activation elicited by fructose feeding, suggesting inhibition of glycation, oxidative stress and nuclear factor-κB activation may be involved in the antifibrotic mechanisms. Betanin also showed anitglycative effect in BSA/fructose system, which supported that anitglycation was involved in betanin's protective roles in vivo. Taken together, the potential for using betanin as an auxillary therapy for diabetic cardiomyopathy deserves to be explored further. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Application of Collagen Scaffold in Tissue Engineering: Recent Advances and New Perspectives

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Chanjuan Dong

2016-02-01

Full Text Available Collagen is the main structural protein of most hard and soft tissues in animals and the human body, which plays an important role in maintaining the biological and structural integrity of the extracellular matrix (ECM and provides physical support to tissues. Collagen can be extracted and purified from a variety of sources and offers low immunogenicity, a porous structure, good permeability, biocompatibility and biodegradability. Collagen scaffolds have been widely used in tissue engineering due to these excellent properties. However, the poor mechanical property of collagen scaffolds limits their applications to some extent. To overcome this shortcoming, collagen scaffolds can be cross-linked by chemical or physical methods or modified with natural/synthetic polymers or inorganic materials. Biochemical factors can also be introduced to the scaffold to further improve its biological activity. This review will summarize the structure and biological characteristics of collagen and introduce the preparation methods and modification strategies of collagen scaffolds. The typical application of a collagen scaffold in tissue engineering (including nerve, bone, cartilage, tendon, ligament, blood vessel and skin will be further provided. The prospects and challenges about their future research and application will also be pointed out.