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Sample records for cell scaffold products

  1. The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    Chen Liu; Caihong Zhu; Jun Li; Pinghui Zhou; Min Chen; Huilin Yang; Bin Li

    2015-01-01

    Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration;however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

  2. In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yan-hong; YANG Qiang; XIA Qun; PENG Jiang; LU Shi-bi; GUO Quan-yi; MA Xin-long

    2013-01-01

    Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM),micro-computed tomography (micro-CT),histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was (708.2±44.7)μg/mg,with GAG (254.7±25.9) μg/mg.Mechanical testing showed the compression moduli (E) were (1.226±0.288) and (0.052±0.007) MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained

  3. Cell-derived matrix coatings for polymeric scaffolds.

    Science.gov (United States)

    Decaris, Martin L; Binder, Bernard Y; Soicher, Matthew A; Bhat, Archana; Leach, J Kent

    2012-10-01

    Cells in culture deposit a complex extracellular matrix that remains intact following decellularization and possesses the capacity to modulate cell phenotype. The direct application of such decellularized matrices (DMs) to 3D substrates is problematic, as transport issues influence the homogeneous deposition, decellularization, and modification of DM surface coatings. In an attempt to address this shortcoming, we hypothesized that DMs deposited by human mesenchymal stem cells (MSCs) could be transferred to the surface of polymeric scaffolds while maintaining their capacity to direct cell fate. The ability of the transferred DM (tDM)-coated scaffolds to enhance the osteogenic differentiation of undifferentiated and osteogenically induced MSCs under osteogenic conditions in vitro was confirmed. tDM-coated scaffolds increased MSC expression of osteogenic marker genes (BGLAP, IBSP) and intracellular alkaline phosphatase production. In addition, undifferentiated MSCs deposited significantly more calcium when seeded onto tDM-coated scaffolds compared with control scaffolds. MSC-seeded tDM-coated scaffolds subcutaneously implanted in nude rats displayed significantly higher blood vessel density after 2 weeks compared with cells on uncoated scaffolds, but we did not observe significant differences in mineral deposition after 8 weeks. These data demonstrate that DM-coatings produced in 2D culture can be successfully transferred to 3D substrates and retain their capacity to modulate cell phenotype.

  4. Design, fabrication and application of tissue engineering used cells scaffold

    Institute of Scientific and Technical Information of China (English)

    WANG Shenguo; BEI Jianzhong

    2001-01-01

    @@ FUNCTIONS OF CELLS SCAFFOLD IN THE TISSUE ENGINEERINGCell, cells scaffold and the construction of tissue and organ are three main factors for the Tissue Engineering. A main function of cells scaffold in tissue engineering is to provide an environment for cells propagation.

  5. Scaffolds and cells for tissue regeneration: different scaffold pore sizes-different cell effects.

    Science.gov (United States)

    Bružauskaitė, Ieva; Bironaitė, Daiva; Bagdonas, Edvardas; Bernotienė, Eiva

    2016-05-01

    During the last decade biomaterial sciences and tissue engineering have become new scientific fields supplying rising demand of regenerative therapy. Tissue engineering requires consolidation of a broad knowledge of cell biology and modern biotechnology investigating biocompatibility of materials and their application for the reconstruction of damaged organs and tissues. Stem cell-based tissue regeneration started from the direct cell transplantation into damaged tissues or blood vessels. However, it is difficult to track transplanted cells and keep them in one particular place of diseased organ. Recently, new technologies such as cultivation of stem cell on the scaffolds and subsequently their implantation into injured tissue have been extensively developed. Successful tissue regeneration requires scaffolds with particular mechanical stability or biodegradability, appropriate size, surface roughness and porosity to provide a suitable microenvironment for the sufficient cell-cell interaction, cell migration, proliferation and differentiation. Further functioning of implanted cells highly depends on the scaffold pore sizes that play an essential role in nutrient and oxygen diffusion and waste removal. In addition, pore sizes strongly influence cell adhesion, cell-cell interaction and cell transmigration across the membrane depending on the various purposes of tissue regeneration. Therefore, this review will highlight contemporary tendencies in application of non-degradable scaffolds and stem cells in regenerative medicine with a particular focus on the pore sizes significantly affecting final recover of diseased organs.

  6. A Transient Cell-shielding Method for Viable MSC Delivery Within Hydrophobic Scaffolds Polymerized in situ

    Science.gov (United States)

    2015-03-27

    A transient cell-shielding method for viable MSC delivery within hydrophobic scaffolds polymerized in situ Ruijing Guo a, b, Catherine L. Ward c...SUBTITLE A transient cell-shielding method for viable MSC delivery within hydrophobic scaffolds polymerized in situ 5a. CONTRACT NUMBER 5b. GRANT...To overcome cell death caused by reaction products from in situ polymerization , we encapsu- lated bone marrow-derived stem cells (BMSCs) in

  7. Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis.

    Science.gov (United States)

    Nakayama, Karina H; Hong, Guosong; Lee, Jerry C; Patel, Jay; Edwards, Bryan; Zaitseva, Tatiana S; Paukshto, Michael V; Dai, Hongjie; Cooke, John P; Woo, Y Joseph; Huang, Ngan F

    2015-07-28

    The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

  8. Human amniotic epithelial cells combined with silk ifbroin scaffold in the repair of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Ting-gang Wang; Jie Xu; Ai-hua Zhu; Hua Lu; Zong-ning Miao; Peng Zhao; Guo-zhen Hui; Wei-jiangWu

    2016-01-01

    Treatment and functional reconstruction atfer central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artiifcial scaffold materials, such as ifbroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithe-lial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk ifbroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk ifbroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inlfammatory cell inifltration at the trans-plant site, milder host-versus-gratf reaction, and a marked improvement in motor function. hTese ifndings conifrm that the transplantation of amniotic epithelial cells combined with silk ifbroin scaffold can promote the repair of spinal cord injury. Silk ifbroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

  9. Cell Invasion in Collagen Scaffold Architectures Characterized by Percolation Theory.

    Science.gov (United States)

    Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G; Best, Serena M; Cameron, Ruth E

    2015-06-24

    The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissue cells reveals this technique as highly relevant for determining the extent of cell invasion.

  10. Influence of scaffold design on 3D printed cell constructs.

    Science.gov (United States)

    Souness, Auryn; Zamboni, Fernanda; Walker, Gavin M; Collins, Maurice N

    2017-02-14

    Additive manufacturing is currently receiving significant attention in the field of tissue engineering and biomaterial science. The development of precise, affordable 3D printing technologies has provided a new platform for novel research to be undertaken in 3D scaffold design and fabrication. In the past, a number of 3D scaffold designs have been fabricated to investigate the potential of a 3D printed scaffold as a construct which could support cellular life. These studies have shown promising results; however, few studies have utilized a low-cost desktop 3D printing technology as a potential rapid manufacturing route for different scaffold designs. Here six scaffold designs were manufactured using a Fused deposition modeling, a "bottom-up" solid freeform fabrication approach, to determine optimal scaffold architecture for three-dimensional cell growth. The scaffolds, produced from PLA, are coated using pullulan and hyaluronic acid to assess the coating influence on cell proliferation and metabolic rate. Scaffolds are characterized both pre- and postprocessing using water uptake analysis, mechanical testing, and morphological evaluation to study the inter-relationships between the printing process, scaffold design, and scaffold properties. It was found that there were key differences between each scaffold design in terms of porosity, diffusivity, swellability, and compressive strength. An optimal design was chosen based on these physical measurements which were then weighted in accordance to design importance based on literature and utilizing a design matrix technique. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017.

  11. Molecular mobility of scaffolds' biopolymers influences cell growth.

    Science.gov (United States)

    Podlipec, Rok; Gorgieva, Selestina; Jurašin, Darija; Urbančič, Iztok; Kokol, Vanja; Strancar, Janez

    2014-09-24

    Understanding biocompatibility of materials and scaffolds is one of the main challenges in the field of tissue engineering and regeneration. The complex nature of cell-biomaterial interaction requires extensive preclinical functionality testing by studying specific cell responses to different biomaterial properties, from morphology and mechanics to surface characteristics at the molecular level. Despite constant improvements, a more general picture of biocompatibility is still lacking and tailormade scaffolds are not yet available. The scope of our study was thus the investigation of the correlation of fibroblast cell growth on different gelatin scaffolds with their morphological, mechanical as well as surface molecular properties. The latter were thoroughly investigated via polymer molecular mobility studied by site-directed spin labeling and electron paramagnetic resonance spectroscopy (EPR) for the first time. Anisotropy of the rotational motion of the gelatin side chain mobility was identified as the most correlated quantity with cell growth in the first days after adhesion, while weaker correlations were found with scaffold viscoelasticity and no correlations with scaffold morphology. Namely, the scaffolds with highly mobile or unrestricted polymers identified with the cell growth being five times less efficient (N(cells) = 60 ± 25 mm(-2)) as compared to cell growth on the scaffolds with considerable part of polymers with the restricted rotational motion (N(cells) = 290 ± 25 mm(-2)). This suggests that molecular mobility of scaffold components could play an important role in cell response to medical devices, reflecting a new aspect of the biocompatibility concept.

  12. Directionally Solidified Biopolymer Scaffolds: Mechanical Properties and Endothelial Cell Responses

    OpenAIRE

    Meghri, Nichols W.; Donius, Amalie E.; Riblett, Benjamin W.; Martin, Elizabeth J.; Clyne, Alisa Morss; Wegst, Ulrike G.K.

    2010-01-01

    Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As...

  13. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds

    DEFF Research Database (Denmark)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette

    2007-01-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We...... polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer h......MSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small...

  14. Recent Advances in Hydroxyapatite Scaffolds Containing Mesenchymal Stem Cells.

    Science.gov (United States)

    Michel, John; Penna, Matthew; Kochen, Juan; Cheung, Herman

    2015-01-01

    Modern day tissue engineering and cellular therapies have gravitated toward using stem cells with scaffolds as a dynamic modality to aid in differentiation and tissue regeneration. Mesenchymal stem cells (MSCs) are one of the most studied stem cells used in combination with scaffolds. These cells differentiate along the osteogenic lineage when seeded on hydroxyapatite containing scaffolds and can be used as a therapeutic option to regenerate various tissues. In recent years, the combination of hydroxyapatite and natural or synthetic polymers has been studied extensively. Due to the interest in these scaffolds, this review will cover the wide range of hydroxyapatite containing scaffolds used with MSCs for in vitro and in vivo experiments. Further, in order to maintain a progressive scope of the field this review article will only focus on literature utilizing adult human derived MSCs (hMSCs) published in the last three years.

  15. Recent Advances in Hydroxyapatite Scaffolds Containing Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    John Michel

    2015-01-01

    Full Text Available Modern day tissue engineering and cellular therapies have gravitated toward using stem cells with scaffolds as a dynamic modality to aid in differentiation and tissue regeneration. Mesenchymal stem cells (MSCs are one of the most studied stem cells used in combination with scaffolds. These cells differentiate along the osteogenic lineage when seeded on hydroxyapatite containing scaffolds and can be used as a therapeutic option to regenerate various tissues. In recent years, the combination of hydroxyapatite and natural or synthetic polymers has been studied extensively. Due to the interest in these scaffolds, this review will cover the wide range of hydroxyapatite containing scaffolds used with MSCs for in vitro and in vivo experiments. Further, in order to maintain a progressive scope of the field this review article will only focus on literature utilizing adult human derived MSCs (hMSCs published in the last three years.

  16. Three-dimensional polycaprolactone scaffold via needleless electrospinning promotes cell proliferation and infiltration.

    Science.gov (United States)

    Li, Dawei; Wu, Tong; He, Nanfei; Wang, Jing; Chen, Weiming; He, Liping; Huang, Chen; Ei-Hamshary, Hany A; Al-Deyab, Salem S; Ke, Qinfei; Mo, Xiumei

    2014-09-01

    Electrospinning has been widely used in fabrication of tissue engineering scaffolds. Currently, most of the electrospun nanofibers performed like a conventional two-dimensional (2D) membrane, which hindered their further applications. Moreover, the low production rate of the traditional needle-electrospinning (NE) also limited the commercialization. In this article, disc-electrospinning (DE) was utilized to fabricate a three-dimensional (3D) scaffold consisting of porous macro/nanoscale fibers. The morphology of the porous structure was investigated by scanning electron microscopy images, which showed irregular pores of nanoscale spreading on the surface of DE polycaprolactone (PCL) fibers. Protein adsorption assessment illustrated the porous structure could significantly enhance proteins pickup, which was 55% higher than that of solid fiber scaffolds. Fibroblasts were cultured on the scaffold. The results demonstrated that DE fiber scaffold could enhance initial cell attachment. In the 7 days of culture, fibroblasts grew faster on DE fiber scaffold in comparison with solid fiber, solvent cast (SC) film and TCP. Fibroblasts on DE fibers showed a stretched shape and integrated with the porous surface tightly. Cells were also found to migrate into the DE scaffold up to 800μm. Results supported the use of DE PCL fibers as a 3D tissue engineering scaffold in soft tissue regeneration.

  17. Developmental Scaffolding

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio

    2015-01-01

    The concept of scaffolding has wide resonance in several scientific fields. Here we attempt to adopt it for the study of development. In this perspective, the embryo is conceived as an integral whole, comprised of several hierarchical modules as in a recurrent circularity of emerging patterns....... Within the developmental hierarchy, each module yields an inter-level relationship that makes it possible for the scaffolding to mediate the production of selectable variations. Awide range of genetic, cellular and morphological mechanisms allows the scaffolding to integrate these modular variations...... into a functionally coordinate unit. A genetic scaffolding accounts for the inherited invariance of pattern formation during the embryo’s growth. At higher level, cells behave as agents endowed with the capacity to interpret any scaffolding variation as signs. The full hierarchy of a multi-level scaffolding...

  18. Synthetic hydrogels as scaffolds for manipulating endothelium cell behaviors

    OpenAIRE

    2011-01-01

    Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells (ECs) proliferation but also manipulate the behaviors and functions of the ECs. In this review paper, the effect of chemical structure, Young's modulus (E) and zeta potential (ζ) of synthetic hydrogel scaffolds on static cell behaviors, including cell morphology, proliferation, cytoskeleton structure and focal adhesion, and on dynamic cell behaviors, including migration velocity and morphology oscillation, as w...

  19. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

    Science.gov (United States)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette; Li, Haisheng; Zou, Xuenong; Flyvbjerg, Allan; Kassem, Moustapha; Bünger, Cody

    2007-02-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

  20. Osteogenic Capacity of Human Adipose-Derived Stem Cells is Preserved Following Triggering of Shape Memory Scaffolds.

    Science.gov (United States)

    Tseng, Ling-Fang; Wang, Jing; Baker, Richard M; Wang, Guirong; Mather, Patrick T; Henderson, James H

    2016-08-01

    Recent advances in shape memory polymers have enabled the study of programmable, shape-changing, cytocompatible tissue engineering scaffolds. For treatment of bone defects, scaffolds with shape memory functionality have been studied for their potential for minimally invasive delivery, conformal fitting to defect margins, and defect stabilization. However, the extent to which the osteogenic differentiation capacity of stem cells resident in shape memory scaffolds is preserved following programmed shape change has not yet been determined. As a result, the feasibility of shape memory polymer scaffolds being employed in stem cell-based treatment strategies remains unclear. To test the hypothesis that stem cell osteogenic differentiation can be preserved during and following triggering of programmed architectural changes in shape memory polymer scaffolds, human adipose-derived stem cells were seeded in shape memory polymer foam scaffolds or in shape memory polymer fibrous scaffolds programmed to expand or contract, respectively, when warmed to body temperature. Osteogenic differentiation in shape-changing and control scaffolds was compared using mineral deposition, protein production, and gene expression assays. For both shape-changing and control scaffolds, qualitatively and quantitatively comparable amounts of mineral deposition were observed; comparable levels of alkaline phosphatase activity were measured; and no significant differences in the expression of genetic markers of osteogenesis were detected. These findings support the feasibility of employing shape memory in scaffolds for stem cell-based therapies for bone repair.

  1. In Vitro Testing of Scaffolds for Mesenchymal Stem Cell-Based Meniscus Tissue Engineering—Introducing a New Biocompatibility Scoring System

    Directory of Open Access Journals (Sweden)

    Felix P. Achatz

    2016-04-01

    Full Text Available A combination of mesenchymal stem cells (MSCs and scaffolds seems to be a promising approach for meniscus repair. To facilitate the search for an appropriate scaffold material a reliable and objective in vitro testing system is essential. This paper introduces a new scoring for this purpose and analyzes a hyaluronic acid (HA gelatin composite scaffold and a polyurethane scaffold in combination with MSCs for tissue engineering of meniscus. The pore quality and interconnectivity of pores of a HA gelatin composite scaffold and a polyurethane scaffold were analyzed by surface photography and Berliner-Blau-BSA-solution vacuum filling. Further the two scaffold materials were vacuum-filled with human MSCs and analyzed by histology and immunohistochemistry after 21 days in chondrogenic media to determine cell distribution and cell survival as well as proteoglycan production, collagen type I and II content. The polyurethane scaffold showed better results than the hyaluronic acid gelatin composite scaffold, with signs of central necrosis in the HA gelatin composite scaffolds. The polyurethane scaffold showed good porosity, excellent pore interconnectivity, good cell distribution and cell survival, as well as an extensive content of proteoglycans and collagen type II. The polyurethane scaffold seems to be a promising biomaterial for a mesenchymal stem cell-based tissue engineering approach for meniscal repair. The new score could be applied as a new standard for in vitro scaffold testing.

  2. Laser printing of cells into 3D scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ovsianikov, A; Gruene, M; Koch, L; Maiorana, F; Chichkov, B [Nanotechnology Department, Laser Zentrum Hannover eV, Hollerithallee 8, 30419 Hannover (Germany); Pflaum, M; Wilhelmi, M; Haverich, A, E-mail: a.ovsianikov@lzh.d [Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover (Germany)

    2010-03-15

    One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

  3. Production and characterization of chitosan/gelatin/β-TCP scaffolds for improved bone tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Serra, I.R.; Fradique, R.; Vallejo, M.C.S.; Correia, T.R.; Miguel, S.P.; Correia, I.J., E-mail: icorreia@ubi.pt

    2015-10-01

    Recently, bone tissue engineering emerged as a viable therapeutic alternative, comprising bone implants and new personalized scaffolds to be used in bone replacement and regeneration. In this study, biocompatible scaffolds were produced by freeze-drying, using different formulations (chitosan, chitosan/gelatin, chitosan/β-TCP and chitosan/gelatin/β-TCP) to be used as temporary templates during bone tissue regeneration. Sample characterization was performed through attenuated total reflectance-Fourier transform infrared spectroscopy, X-ray diffraction and energy dispersive spectroscopy analysis. Mechanical characterization and porosity analysis were performed through uniaxial compression test and liquid displacement method, respectively. In vitro studies were also done to evaluate the biomineralization activity and the cytotoxic profile of the scaffolds. Scanning electron and confocal microscopy analysis were used to study cell adhesion and proliferation at the scaffold surface and within their structure. Moreover, the antibacterial activity of the scaffolds was also evaluated through the agar diffusion method. Overall, the results obtained revealed that the produced scaffolds are bioactive and biocompatible, allow cell internalization and show antimicrobial activity against Staphylococcus aureus. Such, make these 3D structures as potential candidates for being used on the bone tissue regeneration, since they promote cell adhesion and proliferation and also prevent biofilm development at their surfaces, which is usually the main cause of implant failure. - Highlights: • Production of 3D scaffolds composed by chitosan/gelatin/β-TCP by freeze-drying for bone regeneration • Physicochemical characterization of the bone substitutes by SEM, FTIR, XRD and EDS • Evaluation of the cytotoxic profile and antibacterial activity of the 3D structures through in vitro assays.

  4. Silk scaffolds with tunable mechanical capability for cell differentiation.

    Science.gov (United States)

    Bai, Shumeng; Han, Hongyan; Huang, Xiaowei; Xu, Weian; Kaplan, David L; Zhu, Hesun; Lu, Qiang

    2015-07-01

    Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an "inert" material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high β-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall β-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells toward myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features.

  5. Platinum blue staining of cells grown in electrospun scaffolds.

    Science.gov (United States)

    Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

    2014-01-01

    Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

  6. Molecular Design of Synthetic Biodegradable Polymers as Cell Scaffold Materials

    Institute of Scientific and Technical Information of China (English)

    WANG Shen-guo; WAN Yu-qing; CAI Qing; HE Bin; CHEN Wen-na

    2004-01-01

    Poly(lactic acid) and its copolymers are regarded as the most useful biomaterials. The good biocompatibility, biodegradability and mechanical properties of them make the synthetic biodegradable polymers have primary application to tissue engineering. The advantages and disadvantages of the synthetic biodegradable polymers as cell scaffold materials are evaluated. This article reviews the modification of polylactide-family aliphatic polymers to improve the cell affinity when the polymers are used as cell scaffolds. We have developed four main approaches: to modify polyester cell scaffolds in combination of plasma treating and collagen coating; to introduce hydrophilic segments into aliphatic polyester backbones; to introduce pendant functional groups into polyester chains; to modify polyester with dextran. The results of the cell cultures prove that the approaches mentioned above have improved the cell affinity of the polyesters and have modulated cell function such as adhesion, proliferation and migration.

  7. A 1-min method for homogenous cell seeding in porous scaffolds

    NARCIS (Netherlands)

    Tan, Lijun; Ren, Yijin; Kuijer, Roel

    2012-01-01

    The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure for both calcium phosphate ceramic scaffolds and polymer scaffolds. Poly(D,L-lactic acid) and beta-tri-calcium phosphate scaffolds were seeded with MC3T3-E1 cells in a syringe. Scaffolds were put in the syrin

  8. Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications

    Directory of Open Access Journals (Sweden)

    Kazi M. Zakir Hossain

    2015-07-01

    Full Text Available Tubular scaffolds with aligned polylactic acid (PLA fibres were fabricated for cell guide applications by immersing rolled PLA fibre mats into a polyvinyl acetate (PVAc solution to bind the mats. The PVAc solution was also mixed with up to 30 wt % β-tricalcium phosphate (β-TCP content. Cross-sectional images of the scaffold materials obtained via scanning electron microscopy (SEM revealed the aligned fibre morphology along with a significant number of voids in between the bundles of fibres. The addition of β-TCP into the scaffolds played an important role in increasing the void content from 17.1% to 25.3% for the 30 wt % β-TCP loading, which was measured via micro-CT (µCT analysis. Furthermore, µCT analyses revealed the distribution of aggregated β-TCP particles in between the various PLA fibre layers of the scaffold. The compressive modulus properties of the scaffolds increased from 66 MPa to 83 MPa and the compressive strength properties decreased from 67 MPa to 41 MPa for the 30 wt % β-TCP content scaffold. The scaffolds produced were observed to change into a soft and flexible form which demonstrated shape recovery properties after immersion in phosphate buffered saline (PBS media at 37 °C for 24 h. The cytocompatibility studies (using MG-63 human osteosarcoma cell line revealed preferential cell proliferation along the longitudinal direction of the fibres as compared to the control tissue culture plastic. The manufacturing process highlighted above reveals a simple process for inducing controlled cell alignment and varying porosity features within tubular scaffolds for potential tissue engineering applications.

  9. Cell Invasion in Collagen Scaffold Architectures Characterized by Percolation Theory

    OpenAIRE

    Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G.; Best, Serena M.; Cameron, Ruth E.

    2015-01-01

    This is the final version of the article. It first appeared from Wiley at http://dx.doi.org/10.1002/adhm.201500197. The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissu...

  10. Scaffolding Productive Language Skills through Sociodramatic Play

    Science.gov (United States)

    Galeano, Rebecca

    2011-01-01

    This article reviews how a receptive, bilingual four-year-old increased her Spanish productive-language skills over five weeks as she engaged in Spanish-language play sessions with bilingual peers. The data show her growing participation in group verbal interactions along with her growing production of her weaker language. In addition, a…

  11. Indirect additive manufacturing as an elegant tool for the production of self-supporting low density gelatin scaffolds.

    Science.gov (United States)

    Van Hoorick, Jasper; Declercq, Heidi; De Muynck, Amelie; Houben, Annemie; Van Hoorebeke, Luc; Cornelissen, Ria; Van Erps, Jürgen; Thienpont, Hugo; Dubruel, Peter; Van Vlierberghe, Sandra

    2015-10-01

    The present work describes for the first time the production of self-supporting low gelatin density (additive manufacturing techniques in the abscence of additives, we applied an indirect fused deposition modelling approach. To realize this, we have printed a sacrificial polyester scaffold which supported the hydrogel material during UV crosslinking, thereby preventing hydrogel structure collapse. After complete curing, the polyester scaffold was selectively dissolved leaving behind a porous, interconnective low density gelatin scaffold. Scaffold structural analysis indicated the success of the selected indirect additive manufacturing approach. Physico-chemical testing revealed scaffold properties (mechanical, degradation, swelling) to depend on the applied gelatin concentration and methacrylamide content. Preliminary biocompatibility studies revealed the cell-interactive and biocompatible properties of the materials developed.

  12. Directionally solidified biopolymer scaffolds: Mechanical properties and endothelial cell responses

    Science.gov (United States)

    Meghri, Nicholas W.; Donius, Amalie E.; Riblett, Benjamin W.; Martin, Elizabeth J.; Clyne, Alisa Morss; Wegst, Ulrike G. K.

    2010-07-01

    Vascularization is a primary challenge in tissue engineering. To achieve it in a tissue scaffold, an environment with the appropriate structural, mechanical, and biochemical cues must be provided enabling endothelial cells to direct blood vessel growth. While biochemical stimuli such as growth factors can be added through the scaffold material, the culture medium, or both, a well-designed tissue engineering scaffold is required to provide the necessary local structural and mechanical cues. As chitosan is a well-known carrier for biochemical stimuli, the focus of this study was on structure-property correlations, to evaluate the effects of composition and processing conditions on the three-dimensional architecture and properties of freeze-cast scaffolds; to establish whether freeze-east scaffolds are promising candidates as constructs promoting vascularization; and to conduct initial tissue culture studies with endothelial cells on flat substrates of identical compositions as those of the scaffolds to test whether these are biocompatible and promote cell attachment and proliferation.

  13. Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells.

    Science.gov (United States)

    Bonandrini, Barbara; Figliuzzi, Marina; Papadimou, Evangelia; Morigi, Marina; Perico, Norberto; Casiraghi, Federica; Dipl, Chemistry; Sangalli, Fabio; Conti, Sara; Benigni, Ariela; Remuzzi, Andrea; Remuzzi, Giuseppe

    2014-05-01

    For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Micro-computerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and

  14. Self-assembling hydrogel scaffolds for photocatalytic hydrogen production

    Science.gov (United States)

    Weingarten, Adam S.; Kazantsev, Roman V.; Palmer, Liam C.; McClendon, Mark; Koltonow, Andrew R.; Samuel, Amanda P. S.; Kiebala, Derek J.; Wasielewski, Michael R.; Stupp, Samuel I.

    2014-11-01

    Integration into a soft material of all the molecular components necessary to generate storable fuels is an interesting target in supramolecular chemistry. The concept is inspired by the internal structure of photosynthetic organelles, such as plant chloroplasts, which colocalize molecules involved in light absorption, charge transport and catalysis to create chemical bonds using light energy. We report here on the light-driven production of hydrogen inside a hydrogel scaffold built by the supramolecular self-assembly of a perylene monoimide amphiphile. The charged ribbons formed can electrostatically attract a nickel-based catalyst, and electrolyte screening promotes gelation. We found the emergent phenomenon that screening by the catalyst or the electrolytes led to two-dimensional crystallization of the chromophore assemblies and enhanced the electronic coupling among the molecules. Photocatalytic production of hydrogen is observed in the three-dimensional environment of the hydrogel scaffold and the material is easily placed on surfaces or in the pores of solid supports.

  15. Nano scaffolds and stem cell therapy in liver tissue engineering

    Science.gov (United States)

    Montaser, Laila M.; Fawzy, Sherin M.

    2015-08-01

    Tissue engineering and regenerative medicine have been constantly developing of late due to the major progress in cell and organ transplantation, as well as advances in materials science and engineering. Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of micro environmental signals and novel methods to track and guide transplanted stem cells. A major complication encountered with stem cell therapies has been the failure of injected cells to engraft to target tissues. The application of nanotechnology to stem cell biology would be able to address those challenges. Combinations of stem cell therapy and nanotechnology in tissue engineering and regenerative medicine have achieved significant advances. These combinations allow nanotechnology to engineer scaffolds with various features to control stem cell fate decisions. Fabrication of Nano fiber cell scaffolds onto which stem cells can adhere and spread, forming a niche-like microenvironment which can guide stem cells to proceed to heal damaged tissues. In this paper, current and emergent approach based on stem cells in the field of liver tissue engineering is presented for specific application. The combination of stem cells and tissue engineering opens new perspectives in tissue regeneration for stem cell therapy because of the potential to control stem cell behavior with the physical and chemical characteristics of the engineered scaffold environment.

  16. Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Changcan; Yuan, Wenjie; Khan, Musammir; Li, Qian [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@tju.edu.cn [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin) Tianjin 300072 (China); Yao, Fanglian [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Tianjin 300072 (China); Zhang, Wencheng, E-mail: wenchengzhang@yahoo.com [Department of Physiology and Pathophysiology, Logistics University of Chinese People' s Armed Police Force, Tianjin 300162 (China)

    2015-05-01

    Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds. - Graphical abstract: PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatin onto the surface of aminated PCU scaffolds (method a). To increase the immobilization amount of gelatin, PEGMAs were grafted onto the scaffold surface by SI-ATRP. PCU-g-PEGMA-g-gelatin scaffolds were prepared by method b. The gelatin modified scaffolds exhibited high hydrophilicity, low platelet adhesion, perfect anti-hemolytic activity, and excellent cell adhesion and proliferation capacity. They might have potential applications as tissue engineering scaffolds for artificial blood vessels. - Highlights: • Hydrophilic scaffolds were prepared by grafting PEGMA and immobilization of gelatins. • Grafting PEGMA enhanced the immobilization amount of gelatin

  17. PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies

    Energy Technology Data Exchange (ETDEWEB)

    Milovac, Dajana, E-mail: dmilovac@fkit.hr [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia); Gamboa-Martínez, Tatiana C. [Centre for Biomaterials and Tissue Engineering, Universitat Politècnica de València (Spain); Ivankovic, Marica [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia); Gallego Ferrer, Gloria [Centre for Biomaterials and Tissue Engineering, Universitat Politècnica de València (Spain); Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine, Valencia (Spain); Ivankovic, Hrvoje [Faculty of Chemical Engineering and Technology, University of Zagreb (Croatia)

    2014-09-01

    In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. - Highlights: • Hydroxyapatite/poly(ε-caprolactone) scaffold with interconnected pores was prepared • Cytotoxicity test showed that the scaffold was not cytotoxic towards MC3T3-E1 cells • The scaffold supported the attachment, proliferation and differentiation of cells • A 3D cell colonization was confirmed using the fluorescence microscopy • The scaffold might be a promising candidate for bone tissue engineering.

  18. Geometry-driven cell organization determines tissue growths in scaffold pores: consequences for fibronectin organization.

    Directory of Open Access Journals (Sweden)

    Pascal Joly

    Full Text Available To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM. Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1 a simple geometric description predicts cellular organization during pore filling at the cell level and that 2 pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01 and reduced once the pores were closed (ρ = 0.26±0.04 indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.

  19. Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

    Science.gov (United States)

    Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

    2007-01-01

    Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and

  20. Effects of sterilisation method on surface topography and in-vitro cell behaviour of electrostatically spun scaffolds.

    Science.gov (United States)

    Andrews, Kirstie D; Hunt, John A; Black, Richard A

    2007-02-01

    Electrostatic spinning is a potentially significant technique for scaffold production within the field of tissue engineering; however, the effect of sterilisation upon these structures is not known. This research investigated the extent of any topographical alteration to electrostatically spun scaffolds post-production through sterilisation, and examined any subsequent effect on contacting cells. Scaffolds made from Tecoflex SG-80A polyurethane were sterilised using ethylene oxide and UV-ozone. Scaffold topography was characterized in terms of inter-fibre separation (ifs), fibre diameter (f.dia) and surface roughness. Cell culture was performed over 7 days with both mouse L929 and human embryonic lung fibroblasts, the results of which were assessed using SEM, image analysis and confocal microscopy. Sterilisation by UV-ozone and ethylene oxide decreased ifs and increased f.dia; surface roughness was decreased by UV-ozone but increased by ethylene oxide. Possible mechanisms to explain these observations are discussed, namely photo-oxidative degradation in the case of UV-ozone and process-induced changes in surface roughness. UV-ozone sterilised scaffolds showed greater cell coverage than those treated with ethylene oxide, but lower coverage than all the controls. Changes in cell attachment and morphology were thought to be due to the changes in topography brought about by the sterilisation process. We conclude that surface modification by sterilisation could prove to be a useful tool at the final stage of scaffold production to enhance cell contact, phenotype or function.

  1. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Directory of Open Access Journals (Sweden)

    Chih-Hao Chang

    Full Text Available Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP techniques. A self-developed 3D printer with laser-aided gelling (LAG process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w. Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  2. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity.

    Science.gov (United States)

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity.

  3. 3D Printing Bioceramic Porous Scaffolds with Good Mechanical Property and Cell Affinity

    Science.gov (United States)

    Chang, Chih-Hao; Lin, Chih-Yang; Liu, Fwu-Hsing; Chen, Mark Hung-Chih; Lin, Chun-Pin; Ho, Hong-Nerng; Liao, Yunn-Shiuan

    2015-01-01

    Artificial bone grafting is widely used in current orthopedic surgery for bone defect problems. Unfortunately, surgeons remain unsatisfied with the current commercially available products. One of the major complaints is that these products cannot provide sufficient mechanical strength to support the human skeletal structure. In this study, we aimed to develop a bone scaffold with better mechanical property and good cell affinity by 3D printing (3DP) techniques. A self-developed 3D printer with laser-aided gelling (LAG) process was used to fabricate bioceramic scaffolds with inter-porous structures. To improve the mechanical property of the bioceramic parts after heating, CaCO3 was added to the silica ceramic slurry. CaCO3 was blended into a homogenous SiO2-sol dispersion at weight ratios varying from 0/100 to 5/95 to 9/91 (w/w). Bi-component CaCO3/SiO2-sol was prepared as a biocomposite for the 3DP scaffold. The well-mixed biocomposite was used to fabricate the bioceramic green part using the LAG method. The varied scaffolds were sintered at different temperatures ranging from 900 to 1500°C, and the mechanical property was subsequently analyzed. The scaffolds showed good property with the composite ratio of 5:95 CaCO3:SiO2 at a sintering temperature of 1300°C. The compressive strength was 47 MPa, and the porosity was 34%. The topography of the sintered 3DP bioceramic scaffold was examined by SEM, EDS and XRD. The silica bioceramic presented no cytotoxicity and good MG-63 osteoblast-like cell affinity, demonstrating good biocompatibility. Therefore, the new silica biocomposite is viable for fabricating 3DP bone bioceramics with improved mechanical property and good cell affinity. PMID:26618362

  4. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  5. Three-Dimensional Scaffold from Decellularized Human Gingiva for Cell Cultures: Glycoconjugates and Cell Behavior

    Directory of Open Access Journals (Sweden)

    Seyed Ali Banihashem Rad

    2013-01-01

    Full Text Available Objective: We studied both the presence of some carbohydrate compounds in a three-dimensional (3D matrix harvested from human gingiva and the cell behavior in this matrix.Materials and Methods: In this experimental research, in order to prepare 3D scaffolds, human palatal gingival biopsies were harvested and physically decellularized by freeze-thawing and sodium dodecyl sulfate (SDS. The scaffolds were placed within the rings of blastema tissues obtained from a pinna rabbit, in vitro. We evaluated the presence of glycoconjugatesand cellular behavior according to histological, histochemical and spectrophotometry techniques at one, two and three weeks after culture. One-way analysis of variance (ANOVAcomparedthe groups.Results: Extracellular matrix (ECM remained after decellularization of tissue with 1% SDS. Glycoconjugate contents decreased meaningfully at a higher SDS concentration (p<0.0001. After culture of the ECM scaffold with blastema, we observed increased staining of alcian blue, periodic acid-Schiff (PAS and toluidine blue in the scaffold and a number of other migrant cells which was caused by cell penetrationinto the scaffold. Spectrophotometry results showed an increase in glycosaminoglycans (GAGs of the decellularized scaffolds at three weeks after culture.Conclusion: The present study has shown that a scaffold generated from palatal gingival tissue ECM is a suitable substrate for blastema cell migration and activity.This scaffold maypotentially be useful as a biological scaffold in tissue engineering applications.

  6. Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks

    DEFF Research Database (Denmark)

    Canali, Chiara; Mohanty, Soumyaranjan; Heiskanen, Arto

    2015-01-01

    We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity...... serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures....

  7. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)

    2015-10-27

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  8. Improved cell activity on biodegradable photopolymer scaffolds using titanate nanotube coatings

    Energy Technology Data Exchange (ETDEWEB)

    Beke, S., E-mail: szabolcs.beke@iit.it [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Barenghi, R. [IEIIT, National Research Council (CNR), Via De Marini 6, 16149 Genova (Italy); Farkas, B.; Romano, I. [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Kőrösi, L. [Department of Biotechnology, Nanophage Therapy Center, Enviroinvest Corporation, Kertváros u. 2, H-7632 Pécs (Hungary); Scaglione, S. [IEIIT, National Research Council (CNR), Via De Marini 6, 16149 Genova (Italy); Brandi, F. [Nanophysics, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy); Istituto Nazionale di Ottica, CNR, Via G. Moruzzi 1, 56124-Pisa (Italy)

    2014-11-01

    The development of bioactive materials is in the premise of tissue engineering. For several years, surface functionalization of scaffolds has been one of the most promising approaches to stimulate cellular activity and finally improve implant success. Herein, we describe the development of a bioactive composite scaffold composed of a biodegradable photopolymer scaffold and titanate nanotubes (TNTs). The biodegradable photopolymer scaffolds were fabricated by applying mask-projection excimer laser photocuring at 308 nm. TNTs were synthesized and then spin-coated on the porous scaffolds. Upon culturing fibroblast cells on scaffolds, we found that nanotubes coating affects cell viability and proliferation demonstrating that TNT coatings enhance cell growth on the scaffolds by further improving their surface topography. - Highlights: • Biodegradable scaffolds were produced by mask-assisted UV laser photocuring. • Titanate nanotube deposition was carried out without binding compounds or additives. • Titanate nanotube coatings enhanced cell viability and proliferation.

  9. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Han Sun

    2015-01-01

    Full Text Available Objective: The purpose of this study was to review the current status of calcium phosphate (CaP scaffolds combined with bone morphogenetic proteins (BMPs or mesenchymal stem cells (MSCs in the field of bone tissue engineering (BTE. Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions.

  10. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Institute of Scientific and Technical Information of China (English)

    Han Sun; Hui-Lin Yang

    2015-01-01

    Objective:The purpose of this study was to review the current status of calcium phosphate (CaP) scaffolds combined with bone morphogenetic proteins (BMPs) or mesenchymal stem cells (MSCs) in the field of bone tissue engineering (BTE).Date Sources:Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014,with highly regarded older publications also included.The terms BTE,CaP,BMPs,and MSC were used for the literature search.Study Selection:Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved,reviewed,analyzed,and summarized.Results:An ideal BTE product contains three elements:Scaffold,growth factors,and stem cells.CaP-based scaffolds are popular because of their outstanding biocompatibility,bioactivity,and osteoconductivity.However,they lack stiffness and osteoinductivity.To solve this problem,composite scaffolds of CaP with BMPs have been developed.New bone formation by CaP/BMP composites can reach levels similar to those of autografts.CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness.In addition,a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft.Conclusions:Novel BTE products possess remarkable osteoconduction and osteoinduction capacities,and exhibit balanced degradation with osteogenesis.Further work should yield safe,viable,and efficient materials for the repair of bone lesions.

  11. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    Science.gov (United States)

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  12. Design and production of sintered {beta}-tricalcium phosphate 3D scaffolds for bone tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Carlos F.L. [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal); Silva, Abilio P. [Centro de Ciencia e Tecnologia Aeroespaciais, Universidade da Beira Interior, Covilha (Portugal); Lopes, Luis [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal); Pires, Ines [Instituto de Engenharia Mecanica - Lisboa (IDMEC Lisboa/IST/UTL), Avenida Rovisco Pais, 1049-001 Lisboa (Portugal); Correia, Ilidio J., E-mail: icorreia@ubi.pt [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal)

    2012-07-01

    The characteristics of sintered {beta}-tricalcium phosphate ({beta}-TCP) scaffolds produced by 3D printing were studied by means of X-ray diffraction, Scanning Electron Microscopy, Fourier transform infrared spectroscopy, uniaxial compression tests and cytotoxicity tests, using human osteoblast cells. The results reported include details of the {beta}-TCP scaffolds' porosity, density, phase stability, mechanical behavior and cytotoxic profile. Collectively, these properties are fundamental for the future application of these scaffolds as bone substitutes for individualized therapy. Highlights: Black-Right-Pointing-Pointer {beta}-Tricalcium phosphate ({beta}-TCP) 3D scaffolds were produced by rapid prototyping. Black-Right-Pointing-Pointer Scaffold properties were assessed by SEM, FTIR, XRD and by mechanical tests. Black-Right-Pointing-Pointer The cytotoxic profile of the scaffolds was characterized by in vitro assays. Black-Right-Pointing-Pointer Scaffolds have good properties for its application as bone substitutes for individualized therapy.

  13. A polymer scaffold for self-healing perovskite solar cells

    Science.gov (United States)

    Zhao, Yicheng; Wei, Jing; Li, Heng; Yan, Yin; Zhou, Wenke; Yu, Dapeng; Zhao, Qing

    2016-01-01

    Advancing of the lead halide perovskite solar cells towards photovoltaic market demands large-scale devices of high-power conversion efficiency, high reproducibility and stability via low-cost fabrication technology, and in particular resistance to humid environment for long-time operation. Here we achieve uniform perovskite film based on a novel polymer-scaffold architecture via a mild-temperature process. These solar cells exhibit efficiency of up to ~16% with small variation. The unencapsulated devices retain high output for up to 300 h in highly humid environment (70% relative humidity). Moreover, they show strong humidity resistant and self-healing behaviour, recovering rapidly after removing from water vapour. Not only the film can self-heal in this case, but the corresponding devices can present power conversion efficiency recovery after the water vapour is removed. Our work demonstrates the value of cheap, long chain and hygroscopic polymer scaffold in perovskite solar cells towards commercialization.

  14. Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami

    Science.gov (United States)

    2015-01-01

    Scaffolded DNA origami enables the fabrication of a variety of complex nanostructures that promise utility in diverse fields of application, ranging from biosensing over advanced therapeutics to metamaterials. The broad applicability of DNA origami as a material beyond the level of proof-of-concept studies critically depends, among other factors, on the availability of large amounts of pure single-stranded scaffold DNA. Here, we present a method for the efficient production of M13 bacteriophage-derived genomic DNA using high-cell-density fermentation of Escherichia coli in stirred-tank bioreactors. We achieve phage titers of up to 1.6 × 1014 plaque-forming units per mL. Downstream processing yields up to 410 mg of high-quality single-stranded DNA per one liter reaction volume, thus upgrading DNA origami-based nanotechnology from the milligram to the gram scale. PMID:26028443

  15. Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami.

    Science.gov (United States)

    Kick, Benjamin; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

    2015-07-08

    Scaffolded DNA origami enables the fabrication of a variety of complex nanostructures that promise utility in diverse fields of application, ranging from biosensing over advanced therapeutics to metamaterials. The broad applicability of DNA origami as a material beyond the level of proof-of-concept studies critically depends, among other factors, on the availability of large amounts of pure single-stranded scaffold DNA. Here, we present a method for the efficient production of M13 bacteriophage-derived genomic DNA using high-cell-density fermentation of Escherichia coli in stirred-tank bioreactors. We achieve phage titers of up to 1.6 × 10(14) plaque-forming units per mL. Downstream processing yields up to 410 mg of high-quality single-stranded DNA per one liter reaction volume, thus upgrading DNA origami-based nanotechnology from the milligram to the gram scale.

  16. Development of Microspheres Covered with Hydroxyapatite Nanocrystals as Cell Scaffold for Angiogenesis

    Science.gov (United States)

    Iwamoto, Takashi; Terada, Takahiro; Kogai, Yasumichi; Okada, Masahiro; Fujii, Syuji; Furuzono, Tsutomu

    2012-06-01

    We prepared poly(L-lactide-co-glycolide) cell scaffolds coated with hydroxyapatite (HAp) nanocrystals with 50-100 nm in diameter via the Pickering emulsion method. Our cell scaffolds were composed of biodegradable polymers and HAp nanocrystals as a core and shell, respectively. The scaffolds were spherical but displayed uneven shapes when altering a shear speed of homogenization during syntheses. The surface coverage of HAp nanocrystals was examined because the HAp-coating ratio for the scaffolds was an important factor as cell scaffolds in order to enhance cell adhesion. On the basis of scanning electron microscopy observations and thermogravimetric analyses, it was found that the cell scaffolds showed distorted morphologies, and the HAp-coating ratio decreased with increasing the shear speed in the synthesis because shear stress influenced shapes of the scaffolds.

  17. SYNTHETIC HYDROGELS AS SCAFFOLDS FOR MANIPULATING ENDOTHELIUM CELL BEHAVIORS

    Institute of Scientific and Technical Information of China (English)

    Yong-mei Chen; Jing-jing Yang; Yoshihito Osada; Jian Ping Gong

    2011-01-01

    Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells (ECs) proliferation but also manipulate the behaviors and functions of the ECs. In this review paper, the effect of chemical structure, Young’s modulus (E) and zeta potential (ζ) of synthetic hydrogel scaffolds on static cell behaviors, including cell morphology, proliferation,cytoskeleton structure and focal adhesion, and on dynamic cell behaviors, including migration velocity and morphology oscillation, as well as on EC function such as anti-platelet adhesion, are reported. It was found that negatively charged hydrogels, poly(2-acrylamido-2-methylpropanesulfonie sodium) (PNaAMPS) and poly(sodium p-styrene sulphonate) (PNaSS), can directly promote cell proliferation, with no need of surface modification by any cell-adhesive proteins or peptides at the environment of serum-containing medium. In addition, the Young’s modulus (E) and zeta potential (ζ) of hydrogel scaffolds are quantitatively tuned by copolymer hydrogels, poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), in which the two kinds of negatively charged monomers NaAMPS and NaSS are copolymerized with neutral monomer, N,N-dimethylacrylamide (DMAAm). It was found that the critical zeta potential of hydrogels manipulating EC morphology, proliferation, and motility is ζcritical = -20.83 mV and ζcritical = -14.0 mV for poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), respectively. The above mentioned EC behaviors well correlate with the adsorption of fibronectin,a kind of cell-adhesive protein, on the hydrogel surfaces. Furthermore, adhered platelets on the EC monolayers cultured on the hydrogel scaffolds obviously decreases with an increase of the Young’s modulus (E) of the hydrogels, especially when E > 60 kPa. Glycocalyx assay and gene expression of ECs demonstrate that the anti-platelet adhesion well correlates with the EC-specific glycocalyx. The above investigation suggests that understanding the relationship

  18. A scaffold-filter model for studying the chondrogenic differentiation of stem cells in vitro.

    Science.gov (United States)

    Zhang, Ling; Zheng, Li; Fan, Hong S; Zhang, Xing D

    2017-01-01

    This study was undertaken to explore the synergistic effect of scaffold materials and a cartilage-like environment on the chondrogenic differentiation of stem cells. Because stem cells encapsulated in a cartilage scaffold will be induced by scaffold molecules as well as permeable molecules from the surroundings, it is impossible to optimize a chondro-inducible scaffold without considering environmental sensitivity. How do we know if a designed scaffold will be sufficient prior to implantation? In this study, bone marrow mesenchymal stem cells (bMSCs) were seeded in various scaffolds, including collagen hydrogel, collage/sodium alginate hydrogel, collagen sponge and silk fibroin sponge. The cell-scaffold complex was encapsulated in a filter pocket to avoid direct contact with co-cultured chondrocytes. Scaffolds differed in the ability to adsorb inducible molecules expressed by chondrocytes, as evidenced by various expressions of cartilage specific proteins and genes. Collagen hydrogel unexpectedly supported chondrogenic differentiation in an environment filled with chondrocytes secretion better than other reinforced scaffolds, which is consistent with the previous experiment in vivo. This result indicated that the environmental sensitivity of a scaffold is important for in vivo chondro-induction. This in vitro scaffold-filter model may be useful as a precursor to investigate the chondro-inducing potential of various scaffolds for cartilage repair.

  19. Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells.

    Science.gov (United States)

    Alexander, Dorothea; Hoffmann, Jürgen; Munz, Adelheid; Friedrich, Björn; Geis-Gerstorfer, Jürgen; Reinert, Siegmar

    2008-03-01

    For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12-15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.

  20. Effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs

    Science.gov (United States)

    Wang, Zhi; Qing, Quan; Chen, Xi; Liu, Cheng-Jun; Luo, Jing-Cong; Hu, Jin-Lian; Qin, Ting-Wu

    2016-12-01

    The purpose of this study was to investigate the effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs. Tenocytes were obtained from tail tendons of rats. Polydimethylsiloxane (PDMS) was used to fabricate three types of scaffolds with varying surface morphological characteristics, i.e., smooth, micro-grooved, and porous surfaces, respectively. The tenocytes were seeded on the surfaces of the scaffolds to form tenocyte-scaffold constructs. The constructs were cryopreserved in a vitreous cryoprotectant (CPA) with a multi-step protocol. The cell adhesion to scaffolds was observed with electronic scanning microscopy (SEM). The elongation index of the living tenocytes and ratio of live/dead cell number were examined based on a live/dead dual fluorescent staining technique, and the survival rate of tenocytes was studied with flow cytometry (FC). The results showed the shapes of tenocytes varied between the different groups: flat or polygonal (on smooth surface), spindle (on micro-grooved surface), and spindle or ellipse (on porous surface). After thawing, the porous surface got the most living tenocytes and a higher survival rate, suggesting its potential application for vitreous cryopreservation of engineered tendon constructs.

  1. Scaffold-independent Patterning of Cells using Magnetic Nanoparticles

    Science.gov (United States)

    Ghosh, Suvojit; Biswas, Moanaro; Elankumaran, Subbiah; Puri, Ishwar

    2013-03-01

    Spatial patterning of cells in vitro relies on direct contact of cells on to solid surfaces. Scaffold independent patterning of cells has never been achieved so far. Patterning of cells has wide applications including stem cell biology, tissue architecture and regenerative medicine besides fundamental biology. Magnetized cells in a suspension can be manipulated using an externally applied magnetic field enabling directed patterning. We magnetized mammalian cells by internalization of superparamagnetic nanoparticles coated with bovine serum albumin (BSA). A magnetic field is then used to arrange cells in a desired pattern on a substrate or in suspension. The control strategy is derived from the self-assembly of magnetic colloids in a liquid considering magnetostatic interactions. The range of achievable structural features promise novel experimental methods investigating the influence of tissue shape and size on cell population dynamics wherein Fickian diffusion of autocrine growth signals are known to play a significant role. By eliminating the need for a scaffold, intercellular adhesion mechanics and the effects of temporally regulated signals can be investigated. The findings can be applied to novel tissue engineering methods.

  2. Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

    Directory of Open Access Journals (Sweden)

    Hermann Agis

    Full Text Available Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

  3. Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

    Science.gov (United States)

    Agis, Hermann; Collins, Amy; Taut, Andrei D; Jin, Qiming; Kruger, Laura; Görlach, Christoph; Giannobile, William V

    2014-01-01

    Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

  4. Fabrication of Nanostructured Poly-ε-caprolactone 3D Scaffolds for 3D Cell Culture Technology

    KAUST Repository

    Schipani, Rossana

    2015-04-21

    Tissue engineering is receiving tremendous attention due to the necessity to overcome the limitations related to injured or diseased tissues or organs. It is the perfect combination of cells and biomimetic-engineered materials. With the appropriate biochemical factors, it is possible to develop new effective bio-devices that are capable to improve or replace biological functions. Latest developments in microfabrication methods, employing mostly synthetic biomaterials, allow the production of three-dimensional (3D) scaffolds that are able to direct cell-to-cell interactions and specific cellular functions in order to drive tissue regeneration or cell transplantation. The presented work offers a rapid and efficient method of 3D scaffolds fabrication by using optical lithography and micro-molding techniques. Bioresorbable polymer poly-ε-caprolactone (PCL) was the material used thanks to its high biocompatibility and ability to naturally degrade in tissues. 3D PCL substrates show a particular combination in the designed length scale: cylindrical shaped pillars with 10μm diameter, 10μm height, arranged in a hexagonal lattice with spacing of 20μm were obtained. The sidewalls of the pillars were nanostructured by attributing a 3D architecture to the scaffold. The suitability of these devices as cell culture technology supports was evaluated by plating NIH/3T3 mouse embryonic fibroblasts and human Neural Stem Cells (hNSC) on them. Scanning Electron Microscopy (SEM) analysis was carried out in order to examine the micro- and nano-patterns on the surface of the supports. In addition, after seeding of cells, SEM and immunofluorescence characterization of the fabricated systems were performed to check adhesion, growth and proliferation. It was observed that cells grow and develop healthy on the bio-polymeric devices by giving rise to well-interconnected networks. 3D PCL nano-patterned pillared scaffold therefore may have considerable potential as effective tool for

  5. Two-photon polymerization of immune cell scaffolds

    DEFF Research Database (Denmark)

    Olsen, Mark Holm

    and easy to use chip integrated migration platform. Free-form constructs with three-dimensional (3D) microporosity were fabricated by two-photon polymerization inside the closed microchannel of an injection molded commercially available polymer chip for analysis of directed cell migration. Acrylate...... also present a poly (ethylene glycol) diacrylate (PEGDA) based strategy to fabricate soft 3D hydrogel scaffolds. Our experiments with the hydrogel confirm we can control the mechanical properties and introduce biochemical cues on the surface that are recognized by fibroblast cells. Finally we present...

  6. Novel fiber-based pure chitosan scaffold for tendon augmentation: biomechanical and cell biological evaluation.

    Science.gov (United States)

    Nowotny, J; Aibibu, D; Farack, J; Nimtschke, U; Hild, M; Gelinsky, M; Kasten, P; Cherif, Ch

    2016-07-01

    One possibility to improve the mechanical properties after tendon ruptures is augmentation with a scaffold. Based on wet spinning technology, chitosan fibres were processed to a novel pure high-grade multifilament yarn with reproducible quality. The fibres were braided to obtain a 3D tendon scaffold. The CS fibres and scaffolds were evaluated biomechanically and compared to human supraspinatus (SSP) tendons. For the cytobiological characterization, in vitro cell culture experiments with human mesenchymal stem cells (hMSC) were performed. Three types of 3D circular braided scaffolds were fabricated. Significantly, higher ultimate stress values were measured for scaffold with larger filament yarn, compared to scaffold with smaller filament yarn. During cultivation over 28 days, the cells showed in dependence of isolation method and/or donor a doubling or tripling of the cell number or even a six-fold increase on the CS scaffold, which was comparable to the control (polystyrene) or in the case of cells obtained from human biceps tendon even higher proliferation rates. After 14 days, the scaffold surface was covered homogeneously with a cell layer. In summary, the present work demonstrates that braided chitosan scaffolds constitute a straightforward approach for designing tendon analogues, maintaining important flexibility in scaffold design and providing favourable mechanical properties of the resulting construct.

  7. Biodegradable nanocomposite microparticles as drug delivering injectable cell scaffolds.

    Science.gov (United States)

    Wen, Yanhong; Gallego, Monica Ramos; Nielsen, Lene Feldskov; Jorgensen, Lene; Everland, Hanne; Møller, Eva Horn; Nielsen, Hanne Mørck

    2011-11-30

    Injectable cell scaffolds play a dual role in tissue engineering by supporting cellular functions and delivering bioactive molecules. The present study aimed at developing biodegradable nanocomposite microparticles with sustained drug delivery properties thus potentially being suitable for autologous stem cell therapy. Semi-crystalline poly(l-lactide/dl-lactide) (PLDL70) and poly(l-lactide-co-glycolide) (PLGA85) were used to prepare nanoparticles by the double emulsion method. Uniform and spherical nanoparticles were obtained at an average size of 270-300 nm. The thrombin receptor activator peptide-6 (TRAP-6) was successfully loaded in PLDL70 and PLGA85 nanoparticles. During the 30 days' release, PLDL70 nanoparticles showed sustainable release with only 30% TRAP-6 released within the first 15 days, while almost 80% TRAP-6 was released from PLGA85 nanoparticles during the same time interval. The release mechanism was found to depend on the crystallinity and composition of the nanoparticles. Subsequently, mPEG-PLGA nanocomposite microparticles containing PLDL70 nanoparticles were produced by the ultrasonic atomization method and evaluated to successfully preserve the intrinsic particulate properties and the sustainable release profile, which was identical to that of the nanoparticles. Good cell adhesion of the human fibroblasts onto the nanocomposite microparticles was observed, indicating the desired cell biocompatibility. The presented results thus demonstrate the development of nanocomposite microparticles tailored for sustainable drug release for application as injectable cell scaffolds.

  8. Scaffolding Online Collaborative Critiquing for Educational Video Production

    Directory of Open Access Journals (Sweden)

    Yiong Hwee Teo

    2009-03-01

    Full Text Available In art, design and media education, learning from examples has been an established way to coach students. To derive greater benefits, teachers should get students to go beyond mere studying of examples. This paper focuses on engaging novice learners in collaborative critiquing of real examples of professional work and past student work in the context of producing an educational video project. While critiquing of such works is not new in art education, there is however scant literature on how to involve students in collaborative critiquing in an online environment involving video projects. A four-step critique model was therefore designed as procedural scaffolding and implemented in an online system, Knowledge Community. A group of Singapore pre-service teachers were engaged in online collaborative critiquing of videos before they embarked on their video projects to illustrate what constitutes good and bad video production. This research points to the value of online collaborative critiquing as a way to facilitate novice designers‟ progress towards expertise. In this environment learners are able to look at problems through multiple perspectives, generate their own solutions and build knowledge that uses the overlapping expertise of the online community.

  9. Molecular scaffold analysis of natural products databases in the public domain.

    Science.gov (United States)

    Yongye, Austin B; Waddell, Jacob; Medina-Franco, José L

    2012-11-01

    Natural products represent important sources of bioactive compounds in drug discovery efforts. In this work, we compiled five natural products databases available in the public domain and performed a comprehensive chemoinformatic analysis focused on the content and diversity of the scaffolds with an overview of the diversity based on molecular fingerprints. The natural products databases were compared with each other and with a set of molecules obtained from in-house combinatorial libraries, and with a general screening commercial library. It was found that publicly available natural products databases have different scaffold diversity. In contrast to the common concept that larger libraries have the largest scaffold diversity, the largest natural products collection analyzed in this work was not the most diverse. The general screening library showed, overall, the highest scaffold diversity. However, considering the most frequent scaffolds, the general reference library was the least diverse. In general, natural products databases in the public domain showed low molecule overlap. In addition to benzene and acyclic compounds, flavones, coumarins, and flavanones were identified as the most frequent molecular scaffolds across the different natural products collections. The results of this work have direct implications in the computational and experimental screening of natural product databases for drug discovery.

  10. Macrophage interactions with polylactic acid and chitosan scaffolds lead to improved recruitment of human mesenchymal stem/stromal cells: a comprehensive study with different immune cells.

    Science.gov (United States)

    Caires, Hugo R; Esteves, Tiago; Quelhas, Pedro; Barbosa, Mário A; Navarro, Melba; Almeida, Catarina R

    2016-09-01

    Despite the importance of immune cell-biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment.

  11. Novel nanometer scaffolds regulate the biological behaviors of neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Jihui Zhou; Zhiqiang Liu; Fuge Sui; Meng Yao; Yansong Wang; Yugang Liu; Feipeng Tian; Qiang Li; He Xiaofeng; Lin Shao

    2013-01-01

    Ideal tissue-engineered scaffold materials regulate proliferation, apoptosis and differentiation of cells seeded on them by regulating gene expression. In this study, aligned and randomly oriented collagen nanofiber scaffolds were prepared using electronic spinning technology. Their diameters and appearance reached the standards of tissue-engineered nanometer scaffolds. The nanofiber scaffolds were characterized by a high swelling ratio, high porosity and good mechanical properties. The proliferation of spinal cord-derived neural stem cells on novel nanofiber scaffolds was obviously enhanced. The proportions of cells in the S and G2/M phases noticeably increased. Moreover, the proliferation rate of neural stem cells on the aligned collagen nanofiber scaffolds was high. The expression levels of cyclin D1 and cyclin-dependent kinase 2 were increased. Bcl-2 expression was significantly increased, but Bax and caspase-3 gene expressions were obviously decreased. There was no significant difference in the differentiation of neural stem cells into neurons on aligned and randomly oriented collagen nanofiber scaffolds. These results indicate that novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit apoptosis without inducing differentiation. Nanofiber scaffolds regulate apoptosis and proliferation in neural stem cells by altering gene expression.

  12. Geometry optimization of a fibrous scaffold based on mathematical modelling and CFD simulation of a dynamic cell culture

    DEFF Research Database (Denmark)

    Tajsoleiman, Tannaz; J. Abdekhodaie, Mohammad; Gernaey, Krist

    2016-01-01

    simulation of cartilage cell culture under a perfusion flow, which allows not only to characterize the supply of nutrients and metabolic products inside a fibrous scaffold, but also to assess the overall culture condition and predict the cell growth rate. Afterwards, the simulation results supported finding...... an optimized design of the scaffold within a new mathematical optimization algorithm that is proposed. The main concept of this optimization routine isto maintain a large effective surface while simultaneously keeping the shear stress levelin an operating range that is expected to be supporting growth...

  13. Polymeric scaffolds as stem cell carriers in bone repair.

    Science.gov (United States)

    Rossi, Filippo; Santoro, Marco; Perale, Giuseppe

    2015-10-01

    Although bone has a high potential to regenerate itself after damage and injury, the efficacious repair of large bone defects resulting from resection, trauma or non-union fractures still requires the implantation of bone grafts. Materials science, in conjunction with biotechnology, can satisfy these needs by developing artificial bones, synthetic substitutes and organ implants. In particular, recent advances in polymer science have provided several innovations, underlying the increasing importance of macromolecules in this field. To address the increasing need for improved bone substitutes, tissue engineering seeks to create synthetic, three-dimensional scaffolds made from polymeric materials, incorporating stem cells and growth factors, to induce new bone tissue formation. Polymeric materials have shown a great affinity for cell transplantation and differentiation and, moreover, their structure can be tuned in order to maintain an adequate mechanical resistance and contemporarily be fully bioresorbable. This review emphasizes recent progress in polymer science that allows relaible polymeric scaffolds to be synthesized for stem cell growth in bone regeneration.

  14. Characterization and Cell Culture of a Grafted Chitosan Scaffold for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Wen-Chuan Hsieh

    2015-01-01

    Full Text Available Poly(vinyl alcohol (PVA was grafted to chitosan to form a porous scaffold. The PVA-g-chitosan 3D scaffold was then observed by Fourier transform infrared spectroscopy (FT-IR. The water absorbency of PVA-g-chitosan was increased 370% by grafting. Scanning electron microscope (SEM observations of the material revealed that the 3D scaffold is highly porous when formed using a homogenizer at 300 rpm. Compression testing demonstrated that as the amount of chitosan increases, the strength of the 3D scaffold strength reached showed that, by increasing the amount of chitosan, the strength of the 3D scaffold could be increased to 16 × 10−1 MPa. Over 35 days of enzymatic degradation, the 3D scaffold was degraded by various enzymes at rates of up to 10%. In vitro tests showed good cell proliferation and growth in the 3D scaffold.

  15. Fabrication of uniformly cell-laden porous scaffolds using a gas-in-liquid templating technique.

    Science.gov (United States)

    Takei, Takayuki; Aokawa, Ryuta; Shigemitsu, Takamasa; Kawakami, Koei; Yoshida, Masahiro

    2015-11-01

    Design of porous scaffolds in tissue engineering field was challenging. Uniform immobilization of cells in the scaffolds with high porosity was essential for homogeneous tissue formation. The present study was aimed at fabricating uniformly cell-laden porous scaffolds with porosity >74% using the gas-in-liquid foam templating technique. To this end, we used gelatin, microbial transglutaminase and argon gas as a scaffold material, cross-linker of the protein and porogen of scaffold, respectively. We confirmed that a porosity of >74% could be achieved by increasing the gas volume delivered to a gelatin solution. Pore size in the scaffold could be controlled by stirring speed, stirring time and the pore size of the filter through which the gas passed. The foaming technique enabled us to uniformly immobilize a human hepatoblastoma cell line in scaffold. Engraftment efficiency of the cell line entrapped within the scaffold in nude mice was higher than that of cells in free-form. These results showed that the uniformly cell-laden porous scaffolds were promising for tissue engineering.

  16. Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

    Institute of Scientific and Technical Information of China (English)

    GAO Yan; YAN Jing; CUI Xue-jun; WANG Hong-yan; WANG Qing

    2012-01-01

    To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid(PLLA) were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT) assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

  17. Monosaccharide-responsive phenylboronate-polyol cell scaffolds for cell sheet and tissue engineering applications.

    Directory of Open Access Journals (Sweden)

    Rachamalla Maheedhar Reddy

    Full Text Available Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.

  18. A composite demineralized bone matrix--self assembling peptide scaffold for enhancing cell and growth factor activity in bone marrow.

    Science.gov (United States)

    Hou, Tianyong; Li, Zhiqiang; Luo, Fei; Xie, Zhao; Wu, Xuehui; Xing, Junchao; Dong, Shiwu; Xu, Jianzhong

    2014-07-01

    The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic.

  19. Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Carlberg, Bjoern; Liu, Johan [BioNano Systems Laboratory, Department of Microtechnology and Nanoscience, Chalmers University of Technology, Goeteborg, SE-412 96 (Sweden); Axell, Mathilda Zetterstroem; Kuhn, H Georg [Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Goeteborg, SE-413 45 (Sweden); Nannmark, Ulf, E-mail: bjorn.carlberg@chalmers.s, E-mail: mathilda.zetterstrom@neuro.gu.s, E-mail: georg.kuhn@neuro.gu.s, E-mail: ulf.nannmark@anatcell.gu.s, E-mail: jliu@chalmers.s [Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Goeteborg, SE-405 30 (Sweden)

    2009-08-15

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150{mu}m, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1{mu}m. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between

  20. General protocol for the culture of cells on plasma-coated electrospun scaffolds.

    Science.gov (United States)

    Guex, A Géraldine; Fortunato, Giuseppino; Hegemann, Dirk; Tevaearai, Hendrik T; Giraud, Marie-Noëlle

    2013-01-01

    As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.

  1. Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds

    Directory of Open Access Journals (Sweden)

    Chaudhry G. R.

    2004-01-01

    Full Text Available An approach for 3D bone tissue generation from embryonic stem (ES cells was investigated. The ES cells were induced to differentiate into osteogenic precursors, capable of proliferating and subsequently differentiating into bone-forming cells. The differentiated cells and the seeded scaffolds were characterized using von Kossa and Alizarin Red staining, electron microscopy, and RT-PCR analysis. The results demonstrated that ES-derived bone-forming cells attached to and colonized the biocompatible and biodegradable scaffolds. Furthermore, these cells produced bone nodules when grown for 3–4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not an ES cell-specific marker, oct-4. These findings suggest that ES cell can be used for in vitro tissue engineering and cultivation of graftable skeletal structures.

  2. Selective functionalization of nanofiber scaffolds to regulate salivary gland epithelial cell proliferation and polarity.

    Science.gov (United States)

    Cantara, Shraddha I; Soscia, David A; Sequeira, Sharon J; Jean-Gilles, Riffard P; Castracane, James; Larsen, Melinda

    2012-11-01

    Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.

  3. Cell-matrix mechanical interaction in electrospun polymeric scaffolds for tissue engineering: Implications for scaffold design and performance.

    Science.gov (United States)

    Kennedy, Kelsey M; Bhaw-Luximon, Archana; Jhurry, Dhanjay

    2017-03-01

    Engineered scaffolds produced by electrospinning of biodegradable polymers offer a 3D, nanofibrous environment with controllable structural, chemical, and mechanical properties that mimic the extracellular matrix of native tissues and have shown promise for a number of tissue engineering applications. The microscale mechanical interactions between cells and electrospun matrices drive cell behaviors including migration and differentiation that are critical to promote tissue regeneration. Recent developments in understanding these mechanical interactions in electrospun environments are reviewed, with emphasis on how fiber geometry and polymer structure impact on the local mechanical properties of scaffolds, how altering the micromechanics cues cell behaviors, and how, in turn, cellular and extrinsic forces exerted on the matrix mechanically remodel an electrospun scaffold throughout tissue development. Techniques used to measure and visualize these mechanical interactions are described. We provide a critical outlook on technological gaps that must be overcome to advance the ability to design, assess, and manipulate the mechanical environment in electrospun scaffolds toward constructs that may be successfully applied in tissue engineering and regenerative medicine.

  4. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Directory of Open Access Journals (Sweden)

    Anthony Finoli

    2016-01-01

    Full Text Available Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  5. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells.

    Science.gov (United States)

    Finoli, Anthony; Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  6. Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering.

    Science.gov (United States)

    Park, Hyun-Ji; Yu, Seung Jung; Yang, Kisuk; Jin, Yoonhee; Cho, Ann-Na; Kim, Jin; Lee, Bora; Yang, Hee Seok; Im, Sung Gap; Cho, Seung-Woo

    2014-12-01

    Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.

  7. Computer-aided design of microvasculature systems for use in vascular scaffold production

    Energy Technology Data Exchange (ETDEWEB)

    Mondy, William Lafayette [Department of Chemical and Biomedical Engineering, University of South Florida, FL (United States); Cameron, Don [Department of Pathology and Cell Biology, College of Medicine, University of South Florida, FL (United States); Timmermans, Jean-Pierre [Department of Veterinary Sciences, University of Antwerp (Belgium); De Clerck, Nora [Department of Biomedical Sciences University of Antwerp (Belgium); Sasov, Alexander [Skyscan (Belgium); Casteleyn, Christophe [College of Veterinary Medicine, Ghent University (Belgium); Piegl, Les A [Department of Computer Science and Engineering, University of South Florida, FL (United States)

    2009-09-15

    In vitro biomedical engineering of intact, functional vascular networks, which include capillary structures, is a prerequisite for adequate vascular scaffold production. Capillary structures are necessary since they provide the elements and compounds for the growth, function and maintenance of 3D tissue structures. Computer-aided modeling of stereolithographic (STL) micro-computer tomographic (micro-CT) 3D models is a technique that enables us to mimic the design of vascular tree systems containing capillary beds, found in tissues. In our first paper (Mondy et al 2009 Tissue Eng. at press), using micro-CT, we studied the possibility of using vascular tissues to produce data capable of aiding the design of vascular tree scaffolding, which would help in the reverse engineering of a complete vascular tree system including capillary bed structures. In this paper, we used STL models of large datasets of computer-aided design (CAD) data of vascular structures which contained capillary structures that mimic those in the dermal layers of rabbit skin. Using CAD software we created from 3D STL models a bio-CAD design for the development of capillary-containing vascular tree scaffolding for skin. This method is designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantation therapy. Our successful approach to in vitro vasculogenesis will allow the bioengineering of various other types of 3D tissue structures, and as such greatly expands the potential applications of biomedical engineering technology into the fields of biomedical research and medicine.

  8. The growth of stem cells within {beta}-TCP scaffolds in a fluid-dynamic environment

    Energy Technology Data Exchange (ETDEWEB)

    Xu Shanglong [School of Mechatronics Engineering, University of Electronic Science and Technology, Chengdu (China); State Key Laboratory of Mechanical Manufacture System Engineering, Xi' an Jiaotong University, Xi' an (China); Li Dichen [State Key Laboratory of Mechanical Manufacture System Engineering, Xi' an Jiaotong University, Xi' an (China)], E-mail: dcli@mail.xjtu.edu.cn; Xie Youzhuan; Lu Jianxi; Dai Kerong [Department of Orthopaedic Surgery, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

    2008-01-10

    A three-dimensional dynamic perfusion system was developed to provide mass transport and nutrient supply to permit the cell proliferation during the long-term culture inside a {beta}-tricalcium phosphate ({beta}-TCP) scaffold. Also the flow field throughout the scaffold was studied. The porous cylindrical scaffold with a central channel was seeded with the sheep mesenchymal stem cells (MSCs). Then the cell-seeded scaffolds were continuously perfused with the complete {alpha}-MEM medium by a peristaltic pump for 7, 14 and 28 days, respectively. Histological study showed that the cell proliferation rates were different throughout the whole scaffolds and the different cell coverage was shown in different positions of the scaffold. Unoccupied spaces were found in many macropores. A computational fluid dynamics (CFD) modeling was used to simulate the flow conditions within perfused cell-seeded scaffolds to give an insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.52 mm/s) and shear stress (approximately 0.0055 Pa) were found to correspond to increased cell proliferation within the cell-scaffold constructs. Flow speeds were between 0.25 and 0.75 mm/s and shear stresses were between 0.003 and 0.008 Pa in approximately 75% of the regions. This method exhibits novel capabilities to compare the results obtained for different perfusion rates or different scaffold microarchitectures. It may allow specific fluid velocities and shear stresses to be determined to optimize the perfusion flow rate, porous scaffold architecture and distribution of in vitro tissue growth.

  9. Development of Collagen/Demineralized Bone Powder Scaffolds and Periosteum-Derived Cells for Bone Tissue Engineering Application

    Directory of Open Access Journals (Sweden)

    Wilairat Leeanansaksiri

    2013-01-01

    Full Text Available The aim of this study was to investigate physical and biological properties of collagen (COL and demineralized bone powder (DBP scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75–125 µm, 125–250 µm, and 250–500 µm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells, osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250–500 mm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250–500 mm particle size could be considered a potential bone tissue engineering implant.

  10. Influence of Different Three-Dimensional Open Porous Titanium Scaffold Designs on Human Osteoblasts Behavior in Static and Dynamic Cell Investigations

    Directory of Open Access Journals (Sweden)

    Jana Markhoff

    2015-08-01

    Full Text Available In the treatment of osseous defects micro-structured three-dimensional materials for bone replacement serve as leading structure for cell migration, proliferation and bone formation. The scaffold design and culture conditions are crucial for the limited diffusion distance of nutrients and oxygen. In static culture, decreased cell activity and irregular distribution occur within the scaffold. Dynamic conditions entail physical stimulation and constant medium perfusion imitating physiological nutrient supply and metabolite disposal. Therefore, we investigated the influence of different scaffold configurations and cultivation methods on human osteoblasts. Cells were seeded on three-dimensional porous Ti-6Al-4V scaffolds manufactured with selective laser melting (SLM or electron beam melting (EBM varying in porosity, pore size and basic structure (cubic, diagonal, pyramidal and cultured under static and dynamic conditions. Cell viability, migration and matrix production were examined via mitochondrial activity assay, fluorescence staining and ELISA. All scaffolds showed an increasing cell activity and matrix production under static conditions over time. Expectations about the dynamic culture were only partially fulfilled, since it enabled proliferation alike the static one and enhanced cell migration. Overall, the SLM manufactured scaffold with the highest porosity, small pore size and pyramidal basic structure proved to be the most suitable structure for cell proliferation and migration.

  11. Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

    Institute of Scientific and Technical Information of China (English)

    Arash; Khojasteh; Saeed; Reza; Motamedian; Maryam; Rezai; Rad; Mehrnoosh; Hasan; Shahriari; Nasser; Nadjmi

    2015-01-01

    AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells(h DPSCs) on four commercially available scaffold biomaterials. METHODS: hD PSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. h DPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureO ss(Allograft), Cerabone(Xenograft), PLLA(Synthetic), and OSTEON Ⅱ Collagen(Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy(SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase(ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except Sure Oss(Allograft) supported h DPSC adhesion, proliferation and differentiation. hD PSCs seeded on PLLA(Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hD PSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA(Synthetic) and OSTEON ⅡCollagen(Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone(Xenograft) and OSTEON Ⅱ Collagen(Composite) scaffolds, the h DPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hD PSCs. Hence, it may be useful in combination with hD PSCs for cell-based reconstructive therapy.

  12. Graphene derivative scaffolds facilitate in vitro cell survival and maturation of dopaminergic SN4741 cells

    OpenAIRE

    Rodriguez-Losada, Noela; Wendelbo, Rune; Garcia-fernandez, Maria; Pavia, Jose; Martinez-Montañez, Elisa; Lara Muñoz, José Pablo; Arenas, Ernest; Aguirre, José A.

    2014-01-01

    The emerging carbon nanomaterial Graphene (G), in the form of scaffold structure, has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells, providing a promising approach for neural regeneration. We propose the use of G as a scaffold to re-address the dopaminergic (DA) neurons and the residual axons from dead or apoptotic DA neurons in Parkinson´s disease (PD). G could act as a physical support to promote the axonal sprout as a “decele...

  13. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration.

    Science.gov (United States)

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J

    2013-08-01

    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained.

  14. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Mao; Mohanty, Pravansu; Ghosh, Gargi, E-mail: gargi@umich.edu

    2014-11-01

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds.

  15. Stem Cells and Scaffolds for Vascularizing Engineered Tissue Constructs

    Science.gov (United States)

    Luong, E.; Gerecht, S.

    The clinical impact of tissue engineering depends upon our ability to direct cells to form tissues with characteristic structural and mechanical properties from the molecular level up to organized tissue. Induction and creation of functional vascular networks has been one of the main goals of tissue engineering either in vitro, for the transplantation of prevascularized constructs, or in vivo, for cellular organization within the implantation site. In most cases, tissue engineering attempts to recapitulate certain aspects of normal development in order to stimulate cell differentiation and functional tissue assembly. The induction of tissue growth generally involves the use of biodegradable and bioactive materials designed, ideally, to provide a mechanical, physical, and biochemical template for tissue regeneration. Human embryonic stem cells (hESCs), derived from the inner cell mass of a developing blastocyst, are capable of differentiating into all cell types of the body. Specifically, hESCs have the capability to differentiate and form blood vessels de novo in a process called vasculogenesis. Human ESC-derived endothelial progenitor cells (EPCs) and endothelial cells have substantial potential for microvessel formation, in vitro and in vivo. Human adult EPCs are being isolated to understand the fundamental biology of how these cells are regulated as a population and to explore whether these cells can be differentiated and reimplanted as a cellular therapy in order to arrest or even reverse damaged vasculature. This chapter focuses on advances made toward the generation and engineering of functional vascular tissue, focusing on both the scaffolds - the synthetic and biopolymer materials - and the cell sources - hESCs and hEPCs.

  16. Porous chitosan scaffold cross-linked by chemical and natural procedure applied to investigate cell regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Chih-Kai [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Liao, Jiunn-Der, E-mail: jdliao@mail.ncku.edu.tw [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Center of Micro/Nano Science and Technology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Chung, Chia-Wei; Sung, Wei-I. [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Chang, Nai-Jen [Institute of Biomedical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer Polymeric scaffolds, made from chitosan-based films fixed by chemical (citrate) or natural method (genipin), were developed. Black-Right-Pointing-Pointer Nano-indentation with a constant harmonic frequency was applied on porous scaffolds to explore their surface mechanics. Black-Right-Pointing-Pointer The relationship between surface mechanical property and cell-surface interactions of scaffold materials was demonstrated. Black-Right-Pointing-Pointer Porous scaffolds cross-linked by genipin showed adequate cell affinity, non-toxicity, and suitable mechanical properties. - Abstract: Porous chitosan scaffold is used for tissue engineering and drug delivery, but is limited as a scaffold material due to its mechanical weakness, which restrains cell adhesion on the surface. In this study, a chemical reagent (citrate) and a natural reagent (genipin) are used as cross-linkers for the formation of chitosan-based films. Nanoindentation technique with a continuous stiffness measurement system is particularly applied on the porous scaffold surface to examine the characteristic modulus and nanohardness of a porous scaffold surface. The characteristic modulus of a genipin-cross-linked chitosan surface is Almost-Equal-To 2.325 GPa, which is significantly higher than that of an uncross-linked one ( Almost-Equal-To 1.292 GPa). The cell-scaffold surface interaction is assessed. The cell morphology and results of an MTS assay of 3T3-fibroblast cells of a genipin-cross-linked chitosan surface indicate that the enhancement of mechanical properties induced cell adhesion and proliferation on the modified porous scaffold surface. The pore size and mechanical properties of porous chitosan film can be tuned for specific applications such as tissue regeneration.

  17. Comparison of viability of adipose-derived Mesenchymal stem cells on agarose and fibrin glue scaffolds

    Directory of Open Access Journals (Sweden)

    Farzaneh Tafvizi

    2015-06-01

    Full Text Available Background & aim: Utilizing tissue engineering techniques and designing similar structures of the damaged tissues require the use of tools such as scaffolds, cells, and bioactive molecules in vitro. Meanwhile, appropriate cell cultures with the ability to divide and differentiate on the natural scaffolds lacking features like immunogenicity and tumorgenesis is particularly important. Adipose tissue has attracted researchers’ attention due to its abundance of mesenchymal stem cells and its availability through a liposuction. The purpose of the present study was to investigate the reproducibility and viability of the adipose-derived stem cells on natural scaffolds of fibrin glue and agarose. Methods: In the present experimental study, the isolation and identification of the mesenchymal stem cells was performed on tissue obtained from liposuction. The tissues were extensively washed with PBS and were digested with collagenase I, then the mesenchymal stem cells were isolated. The cells were cultured in RPMI medium supplemented with antibiotic. Subsequently, the expression of cell surface markers including CD34, CD44, CD90, and CD105 were analyzed by flow cytometry to confirm the mesenchymal cells. After preparing fibrin glue and agarose scaffolds, the viability and proliferation of the adipose tissue-derived mesenchymal stem cells were examined at the period of 24, 48, and 72 hours by MTT and ELISA assays. The obtained results were analyzed by SPSS ver.19. Results: The results of adipose tissue-derived mesenchymal stem cells culture on the fibrin glue and agarose scaffolds indicated that cell viability on fibrin glue and agarose scaffold were 68.22% and 89.75% in 24 hrs, 64.04% and 66.97% in 48 hours, 222.87% and 1089.68% in 72 hours respectively. Significant proliferation and viability cells on a synthesized agarose scaffold were seen compared to the fibrin glue scaffold after 72 hrs. The viability of the cells significantly increased on the

  18. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    Science.gov (United States)

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  19. Electrospun Biocomposite Polycaprolactone/Collagen Tubes as Scaffolds for Neural Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Joanne M. Hackett

    2010-06-01

    Full Text Available Studies using cellular therapies, scaffolds, and tubular structured implants have been carried out with the goal to restore functional recovery after spinal cord injury (SCI. None of these therapeutic strategies, by themselves, have been shown to be sufficient to achieve complete restoration of function. To reverse the devastating effects of SCI, an interdisciplinary approach that combines materials science and engineering, stem cell biology, and neurosurgery is being carried out. We are currently investigating a scaffold that has the ability to deliver growth factors for the proliferation and differentiation of endogenous stem cells. Neural stem cells (NSCs derived from mice are being used to assess the efficacy of the release of growth factors from the scaffold in vitro. The fabrication of the tubular implant allows a porous scaffold to be formed, which aids in the release of growth factors added to the scaffold.

  20. Architectural and mechanical cues direct mesenchymal stem cell interactions with crosslinked gelatin scaffolds.

    Science.gov (United States)

    McAndrews, Kathleen M; Kim, Min Jeong; Lam, Tuyet Y; McGrail, Daniel J; Dawson, Michelle R

    2014-12-01

    Naturally derived biomaterials have emerged as modulators of cell function and tissue substitutes. Here, we developed crosslinked glutaraldehyde (GTA) scaffolds for the expansion and differentiation of mesenchymal stem cells (MSCs). The mechanical and architectural properties of the scaffolds were altered by varying the concentration of gelatin and GTA. Higher GTA concentrations were associated with an increase in more confined pores and osteogenic differentiation. In addition, myogenic potential varied with crosslinking degree, although bulk mechanical properties were unaltered. Correlation analysis revealed that ALP activity of differentiated MSCs on higher gelatin concentration scaffolds was dependent on traditional effectors, including environment elasticity and spread area. In contrast, the differentiation capacity of cells cultured on lower gelatin concentration scaffolds did not correlate with these factors, instead it was dependent on the hydrated pore structure. These results suggest that scaffold composition can determine what factors direct differentiation and may have critical implications for biomaterial design.

  1. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

    NARCIS (Netherlands)

    Ricci, C.; Mota, C.M.; Moscato, S.; Alessandro, D' D.; Ugel, S.; Sartoris, S.; Bronte, V.; Boggi, U.; Campani, D.; Funel, N.; Moroni, L.; Danti, S.

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol

  2. An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds

    Directory of Open Access Journals (Sweden)

    Alexander Röder

    2015-11-01

    Full Text Available The use of biopolymers as a three dimensional (3D support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1 cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either “low-adhesive” non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.

  3. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone Scaffolds

    Directory of Open Access Journals (Sweden)

    Sònia Palomeras

    2016-04-01

    Full Text Available The cancer stem cell (CSC population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs’ phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone (PCL, a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control. Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

  4. In Vitro Evaluation of Scaffolds for the Delivery of Mesenchymal Stem Cells to Wounds

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Wahl

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis. In vitro results demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCs in vivo. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.

  5. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chih-Hao [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Neurosurgery, Department of Surgery, Kaohsiung Veterans General Hospital, Taiwan, ROC (China); Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Kuo, Shyh Ming [Department of Biomedical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Guei-Sheung [Centre for Eye Research Australia, University of Melbourne (Australia); Chen, Wan-Nan U. [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China); Chuang, Chin-Wen [Department of Electrical Engineering, I-Shou University, Taiwan, ROC (China); Liu, Li-Feng, E-mail: liulf@isu.edu.tw [Department of Biological Science and Technology, I-Shou University, Taiwan, ROC (China)

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. Black-Right-Pointing-Pointer Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. Black-Right-Pointing-Pointer 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 {mu}m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  6. Morphological Effects of HA on the Cell Compatibility of Electrospun HA/PLGA Composite Nanofiber Scaffolds

    Directory of Open Access Journals (Sweden)

    Adnan Haider

    2014-01-01

    Full Text Available Tissue engineering is faced with an uphill challenge to design a platform with appropriate topography and suitable surface chemistry, which could encourage desired cellular activities and guide bone tissue regeneration. To develop such scaffolds, composite nanofiber scaffolds of nHA and sHA with PLGA were fabricated using electrospinning technique. nHA was synthesized using precipitation method, whereas sHA was purchased. The nHA and sHA were suspended in PLGA solution separately and electrospun at optimized electrospinning parameters. The composite nanofiber scaffolds were characterized by FE-SEM, EDX analysis, TEM, XRD analysis, FTIR, and X-ray photoelectron. The potential of the HA/PLGA composite nanofiber as bone scaffolds in terms of their bioactivity and biocompatibility was assessed by culturing the osteoblastic cells onto the composite nanofiber scaffolds. The results from in vitro studies revealed that the nHA/PLGA composite nanofiber scaffolds showed higher cellular adhesion, proliferation, and enhanced osteogenesis performance, along with increased Ca+2 ions release compared to the sHA/PLGA composite nanofiber scaffolds and pristine PLGA nanofiber scaffold. The results show that the structural dependent property of HA might affect its potential as bone scaffold and implantable materials in regenerative medicine and clinical tissue engineering.

  7. Indirect coculture of stem cells with fetal chondrons using PCL electrospun nanofiber scaffolds.

    Science.gov (United States)

    Nikpou, Parisa; Soleimani Rad, Jafar; Mohammad Nejad, Daryoush; Samadi, Nasser; Roshangar, Leila; Navali, Amir Mohammad; Shafaei, Hajar; Nozad Charoudeh, Hojjatollah; Danandeh Oskoei, Neda; Soleimani Rad, Sara

    2017-03-01

    In vitro coculture system provides a powerful tool for tissue engineering. In this study, we evaluated the gene expressions of human adipose-derived stem cells (ASCs) on polycaprolactone (PCL) scaffold in coculture model with fetal chondrons. Electrospun PCL scaffolds (900 nm fiber diameter) were created and human infrapatellar fat pad-adipose-derived stem cells (IPFP-ASCs) were seeded on these scaffolds. Scanning electron microscopy (SEM) showed attachment of human IPFP-ASCs to scaffold. IPFP-ASCs on scaffolds were cocultured with fetal chondrons in transwell. Gene expressions were investigated using real-time polymerase chain reaction (real-time PCR). In comparison with control group, the expression level of collagen type 2 and aggrecan were significantly decreased but Indian Hedgehog(IHH) significantly increased (P fetal chondrons are tending toward osteogenesis rather than chondrogenesis.

  8. Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells

    Science.gov (United States)

    Fang, Yongxiang; Wang, Bin; Zhao, Yannan; Xiao, Zhifeng; Li, Jing; Cui, Yi; Han, Sufang; Wei, Jianshu; Chen, Bing; Han, Jin; Meng, Qingyuan; Hou, Xianglin; Luo, Jianxun; Dai, Jianwu; Jing, Zhizhong

    2017-01-01

    The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs. PMID:28169322

  9. Graphene derivatives as scaffold for ex vivo survival and maturation of dopaminergic SN4741 cells.

    OpenAIRE

    Rodriguez-Losada, Noela; Wendelbo, Rune; Garcia-fernandez, Maria; Pavia, Jose; Martin-Montañez, Elisa; Lara Muñoz, José Pablo; Arenas, Ernest; Aguirre, José A.

    2014-01-01

    Carbon nanomaterial Graphene (G) can form a three-dimensional porous structure with efficient bioconjugation and cell differentiation properties, providing a promising scaffold for neural regeneration. Aims: To study this putative new application of G, we cultured a clonal substantia nigra dopaminergic neuronal progenitor cell line (SN4741) in presence of G as scaffold. Methods: Cells were cultured in DMEM/10% FCS to about 80% confluence and incubated with different concentrations (0.001 to 1...

  10. Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

    Directory of Open Access Journals (Sweden)

    Ketul C. Popat

    2012-11-01

    Full Text Available Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.

  11. Functional stability of endothelial cells on a novel hybrid scaffold for vascular tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Pankajakshan, Divya; Krishnan, Lissy K [Thrombosis Research Unit, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Trivandrum 695 012 (India); Krishnan V, Kalliyana, E-mail: lissykk@sctimst.ac.i [Division of Polymer Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Trivandrum 695 012 (India)

    2010-12-15

    Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation, survival and maintenance of a normal phenotype. In this study we have prepared and evaluated porous poly({epsilon}-caprolactone) (PCL) integrated with fibrin composite (FC) to get a biomimetic hybrid scaffold (FC PCL) with the biological properties of fibrin, fibronectin (FN), gelatin, growth factors and glycosaminoglycans. Reduced platelet adhesion on a human umbilical vein endothelial cell-seeded hybrid scaffold as compared to bare PCL or FC PCL was observed, which suggests the non-thrombogenic nature of the tissue-engineered scaffold. Analysis of real-time polymerase chain reaction (RT-PCR) after 5 days of endothelial cell (EC) culture on a hybrid scaffold indicated that the prothrombotic von Willebrand factor and plasminogen activator inhibitor (PAI) were quiescent and stable. Meanwhile, dynamic expressions of tissue plasminogen activator (tPA) and endothelial nitric oxide synthase indicated the desired cell phenotype on the scaffold. On the hybrid scaffold, shear stress could induce enhanced nitric oxide release, which implicates vaso-responsiveness of EC grown on the tissue-engineered construct. Significant upregulation of mRNA for extracellular matrix (ECM) proteins, collagen IV and elastin, in EC was detected by RT-PCR after growing them on the hybrid scaffold and FC-coated tissue culture polystyrene (FC TCPS) but not on FN-coated TCPS. The results indicate that the FC PCL hybrid scaffold can accomplish a remodeled ECM and non-thrombogenic EC phenotype, and can be further investigated as a scaffold for cardiovascular tissue engineering. (communication)

  12. Development of Composite Poly(Lactide-co-Glycolide)- Nanodiamond Scaffolds for Bone Cell Growth.

    Science.gov (United States)

    Brady, Mariea A; Renzing, Andrea; Douglas, Timothy E L; Liu, Qin; Wille, Sebastian; Parizek, Martin; Bacakova, Lucie; Kromka, Alexander; Jarosova, Marketa; Godier, Greetje; Warnkel, Patrick H

    2015-02-01

    There are relatively few nanotechnologies that can produce nanocomposite scaffolds for cell growth. Electrospinning has emerged as the foremost method of producing nanofibrous biomimetic scaffolds for tissue engineering applications. In this study diamond nanoparticles were integrated into a polymer solution to develop a nanocomposite scaffold containing poly(lactide-co-glycolide) (PLGA) loaded with diamond nanoparticles. To investigate the effect of adding diamond nanoparticles to PLGA scaffolds, primary human mesenchymal stem cells (hMSCs) were seeded on the scaffolds. The cytocompatibility results showed that addition of diamond nanoparticles did not impinge upon cell proliferation, nor was there a cytotoxic cellular response after 9 days in culture. Scanning electron microscopy, transmission electron microscopy, atomic force microscopy and confocal microscopy enabled qualitative characterization of the fibres and revealed cell morphology and number. Furthermore, surface roughness was measured to evaluate diamond nanoparticle modifications, and no significant difference was found between the diamond nanocomposite and pure polymer scaffolds. On the other hand, bright spots on phase images performed by atomic force microscopy suggested a higher hardness at certain points on fibers of the PLGA-nanodiamond composites, which was supported by nanoindentation measurements. This study shows that PLGA nanofibers can be reinforced with nanodiamond without adversely affecting cell behaviour, and thus it sets the foundation for future application of these scaffolds in bone tissue engineering.

  13. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

    Directory of Open Access Journals (Sweden)

    Matschegewski Claudia

    2016-09-01

    Full Text Available The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926. Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation of diminished cell spreading accompanied with reduced cell viability on nonwovens. Among those, highest biocompatibility was assessed for PLLA L214, although being generally low when compared to the planar control surface. Electrospinning was demonstrated as an innovative technique for the fabrication of advanced biomaterials aiming at guided cellular behavior as well as the design of novel implant platforms. A better understanding of cell–biomaterial interactions is desired to further improve implant development.

  14. Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells

    Science.gov (United States)

    Chamieh, Frédéric; Collignon, Anne-Margaux; Coyac, Benjamin R.; Lesieur, Julie; Ribes, Sandy; Sadoine, Jérémy; Llorens, Annie; Nicoletti, Antonino; Letourneur, Didier; Colombier, Marie-Laure; Nazhat, Showan N.; Bouchard, Philippe; Chaussain, Catherine; Rochefort, Gael Y.

    2016-12-01

    Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

  15. Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds.

    Science.gov (United States)

    Gotman, Irena; Ben-David, Dror; Unger, Ronald E; Böse, Thomas; Gutmanas, Elazar Y; Kirkpatrick, C James

    2013-09-01

    Bone tissue regeneration in load-bearing regions of the body requires high-strength porous scaffolds capable of supporting angiogenesis and osteogenesis. 70% porous Nitinol (NiTi) scaffolds with a regular 3-D architecture resembling trabecular bone were produced from Ni foams using an original reactive vapor infiltration technique. The "trabecular Nitinol" scaffolds possessed a high compressive strength of 79 MPa and high permeability of 6.9×10(-6) cm2. The scaffolds were further modified to produce a near Ni-free surface layer and evaluated in terms of Ni ion release and human mesenchymal stem cell (hMSC) proliferation (AlamarBlue), differentiation (alkaline phosphatase activity, ALP) and mineralization (Alizarin Red S staining). Scanning electron microscopy was employed to qualitatively corroborate the results. hMSCs were able to adhere and proliferate on both as-produced and surface-modified trabecular NiTi scaffolds, to acquire an osteoblastic phenotype and produce a mineralized extracellular matrix. Both ALP activity and mineralization were increased on porous scaffolds compared to control polystyrene plates. Experiments in a model coculture system of microvascular endothelial cells and hMSCs demonstrated the formation of prevascular structures in trabecular NiTi scaffolds. These data suggest that load-bearing trabecular Nitinol scaffolds could be effective in regenerating damaged or lost bone tissue.

  16. Desymmetrization of 7-azabicycloalkenes by tandem olefin metathesis for the preparation of natural product scaffolds

    Directory of Open Access Journals (Sweden)

    Deppermann Nina

    2007-12-01

    Full Text Available Abstract Background Tandem olefin metathesis sequences are known to be versatile for the generation of natural product scaffolds and have also been used for ring opening of strained carbo- and heterocycles. In this paper we demonstrate the potential of these reactions for the desymmetrization of 7-azabicycloalkenes. Results We have established efficient protocols for the desymmetrization of different 7-azabicycloalkenes by intra- and intermolecular tandem metathesis sequences with ruthenium based catalysts. Conclusion Desymmetrization of 7-azabicycloalkenes by olefin metathesis is an efficient process for the preparation of common natural product scaffolds such as pyrrolidines, indolizidines and isoindoles.

  17. Desymmetrization of 7-azabicycloalkenes by tandem olefin metathesis for the preparation of natural product scaffolds

    Science.gov (United States)

    Maison, Wolfgang; Büchert, Marina; Deppermann, Nina

    2007-01-01

    Background Tandem olefin metathesis sequences are known to be versatile for the generation of natural product scaffolds and have also been used for ring opening of strained carbo- and heterocycles. In this paper we demonstrate the potential of these reactions for the desymmetrization of 7-azabicycloalkenes. Results We have established efficient protocols for the desymmetrization of different 7-azabicycloalkenes by intra- and intermolecular tandem metathesis sequences with ruthenium based catalysts. Conclusion Desymmetrization of 7-azabicycloalkenes by olefin metathesis is an efficient process for the preparation of common natural product scaffolds such as pyrrolidines, indolizidines and isoindoles. PMID:18088413

  18. Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

    Science.gov (United States)

    He, Min; Storr-Paulsen, Thomas; Wang, Annie L.; Ghezzi, Chiara E.; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P.; Islam, Rakibul; Griffith, May; Islam, M. Mirazul; Hodges, Robin R.; Wnek, Gary E.; Kaplan, David L.; Dartt, Darlene A.

    2016-01-01

    Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study. PMID:27832279

  19. Differentiation of human endometrial stem cells into endothelial-like cells on gelatin/chitosan/bioglass nanofibrous scaffolds.

    Science.gov (United States)

    Shamosi, Atefeh; Mehrabani, Davood; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Siavashi, Vahid; Ghanbari, Hossein; Sharifi, Esmaeel; Roozafzoon, Reza; Ai, Jafar

    2017-02-01

    The capacity of gelatin/chitosan/bioactive glass nanopowders (GEL/CS/BGNPs) scaffolds was investigated for increasing human endometrial stem cells (hEnSCs) differentiation into the endothelial cells in the presence of angiogenic factors. GEL/CS nanofibrous scaffold with different contents of BGNPs were fabricated and assessed. Expression of endothelial markers (CD31, vascular endothelial cadherin (VE-cadherin), and KDR) in differentiated cells was evaluated. Results showed the diameter of nanofiber increases with decreasing the BG content in GEL/CS scaffolds. Moreover, in vitro study indicated that the GEL/CS/BGNPs scaffold with 1.5% BGNPs content provided a suitable three-dimensional structure for endothelial cells differentiation. Thus, the GEL/CS/BGNPs scaffold can be recommended for blood vessels repair.

  20. A surface-modified biodegradable urethral scaffold seeded with urethral epithelial cells

    Institute of Scientific and Technical Information of China (English)

    FU Wei-jun; ZHANG Xu; WANG Zhong-xin; LI Gang; ZHANG Bing-hong; ZHANG Lei; HU Kun; HONG Bao-fa; WANG Xiao-xiong; CUI Fu-zhai

    2011-01-01

    Background Efficient cell adhesion and proliferation is a central issue in cell-based tissue engineering, which offers great promise for repair of urethral defects or strictures. This study evaluated the adhesion and growth of rabbit uroepithelium on a surface-modified three-dimensional poly-L-lactic acid (PLLA) scaffold.Methods Urethral mucosa were harvested from male New Zealand rabbits and the urothelium were dissociated and then cultured. Immunocytochemistry on cultured uroepithelium for pancytokeratin and uroplakin Ⅱ and TE-7 confirmed pure populations. After in vitro proliferation, cells were seeded onto a surface-modified urethral scaffold with non-knitted filaments. The morphology and viability of the cells were examined by immunohistochemical and fluorescence staining.Inverted and scanning microscopes were used to document cell growth and adhesion.Results Three to five days after primary culture, the uroepithelial cells gradually became confluent, assuming a cobblestone pattern. The filaments of the urethral scaffold had excellent biocompatibility and allowed growth of the uroepithelium, without affecting viability. The uroepithelial cells adhered to and grew well on the scaffold. After 3-7 days,the cells grew vigorously and meshes of the scaffold were full of uroepitheliums.Conclusions The surface-modified urethral scaffold with non-knitted filaments allows the growth of uroepithelium and can serve as a carrier for the tissue engineering of urethra.

  1. Large strain stimulation promotes extracellular matrix production and stiffness in an elastomeric scaffold model.

    Science.gov (United States)

    D׳Amore, Antonio; Soares, Joao S; Stella, John A; Zhang, Will; Amoroso, Nicholas J; Mayer, John E; Wagner, William R; Sacks, Michael S

    2016-09-01

    Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the denovo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments.

  2. Direct Mechanical Stimulation of Stem Cells: A Beating Electromechanically Active Scaffold for Cardiac Tissue Engineering.

    Science.gov (United States)

    Gelmi, Amy; Cieslar-Pobuda, Artur; de Muinck, Ebo; Los, Marek; Rafat, Mehrdad; Jager, Edwin W H

    2016-06-01

    The combination of stem cell therapy with a supportive scaffold is a promising approach to improving cardiac tissue engineering. Stem cell therapy can be used to repair nonfunctioning heart tissue and achieve myocardial regeneration, and scaffold materials can be utilized in order to successfully deliver and support stem cells in vivo. Current research describes passive scaffold materials; here an electroactive scaffold that provides electrical, mechanical, and topographical cues to induced human pluripotent stem cells (iPS) is presented. The poly(lactic-co-glycolic acid) fiber scaffold coated with conductive polymer polypyrrole (PPy) is capable of delivering direct electrical and mechanical stimulation to the iPS. The electroactive scaffolds demonstrate no cytotoxic effects on the iPS as well as an increased expression of cardiac markers for both stimulated and unstimulated protocols. This study demonstrates the first application of PPy as a supportive electroactive material for iPS and the first development of a fiber scaffold capable of dynamic mechanical actuation.

  3. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

    Science.gov (United States)

    Morelli, Sabrina; Salerno, Simona; Holopainen, Jani; Ritala, Mikko; De Bartolo, Loredana

    2015-06-20

    The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

  4. Synthesis and Characterization of Carboxymethylcellulose-Methacrylate Hydrogel Cell Scaffolds

    Directory of Open Access Journals (Sweden)

    Andreia Ribeiro

    2010-08-01

    Full Text Available Many carbohydrates pose advantages for tissue engineering applications due to their hydrophilicity, degradability, and availability of chemical groups for modification. For example, carboxymethylcellulose (CMC is a water-soluble cellulose derivative that is degradable by cellulase. Though this enzyme is not synthesized by mammalian cells, cellulase and the fragments derived from CMC degradation are biocompatible. With this in mind, we created biocompatible, selectively degradable CMC-based hydrogels that are stable in routine culture, but degrade when exposed to exogenous cellulase. Solutions of CMC-methacrylate and polyethylene glycol dimethacrylate (PEG-DM were co-crosslinked to form stable hydrogels; we found that greater CMC-methacrylate content resulted in increased gel swelling, protein diffusion and rates of degradation by cellulase, as well as decreased gel shear modulus. CMC-methacrylate/PEG-DM gels modified with the adhesive peptide RGD supported fibroblast adhesion and viability. We conclude that hydrogels based on CMC-methacrylate are suitable for bioengineering applications where selective degradability may be favorable, such as cell scaffolds or controlled release devices.

  5. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    Science.gov (United States)

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  6. Three-Dimensional Stiff Graphene Scaffold on Neural Stem Cells Behavior.

    Science.gov (United States)

    Ma, Qinqin; Yang, Lingyan; Jiang, Ziyun; Song, Qin; Xiao, Miao; Zhang, Dong; Ma, Xun; Wen, Tieqiao; Cheng, Guosheng

    2016-12-21

    Physical cues of the scaffolds, elasticity, and stiffness significantly guide adhesion, proliferation, and differentiation of stem cells. In addressable microenvironments constructed by three-dimensional graphene foams (3D-GFs), neural stem cells (NSCs) interact with and respond to the structural geometry and mechanical properties of porous scaffolds. Our studies aim to investigate NSC behavior on the various stiffness of 3D-GFs. Two kinds of 3D-GFs scaffolds present soft and stiff properties with elasticity moduli of 30 and 64 kPa, respectively. Stiff scaffold enhanced NSC attachment and proliferation with vinculin and integrin gene expression were up-regulated by 2.3 and 1.5 folds, respectively, compared with the soft one. Meanwhile, up-regulated Ki67 expression and almost no variation of nestin expression in a group of the stiff scaffold were observed, implying that the stiff substrate fosters NSC growth and keeps the cells in an active stem state. Furthermore, NSCs grown on stiff scaffold exhibited enhanced differentiation to astrocytes. Interestingly, differentiated neurons on stiff scaffold are suppressed since growth associated protein-43 expression was significantly improved by 5.5 folds.

  7. Novel three-dimensional nerve tissue engineering scaffolds and its biocompatibility with Schwann cells

    Institute of Scientific and Technical Information of China (English)

    YUAN Jian-dong; NIE Wen-bo; FU Qiang; LIAN Xiao-feng; HOU Tie-sheng; TAN Zhi-qing

    2009-01-01

    To develop a novel scaffolding method for the copolymers poly lactide-co-glycolide acid (PLGA) to construct a three-dimensional (3-D) scaffold and explore its biocompatibility through culturing Schwann cells (SCs) on it. Methods: The 3-D scaffolds were made by means of melt spinning, extension and weaving. The queueing disci-pline of the micro-channels were observed under a scan-ning electronic microscope (SEM).The sizes of the micropores and the factors of porosity were also measured. Sciatic nerves were harvested from 3-day-old Sprague Dawley (SD) rats for culture of SCs. SCs were separated, purified, and then implanted on PLGA scaffolds, gelatin sponge and poly-L-lysine (PLL)-coated tissue culture poly-styrene (TCPS) were used as biomaterial and cell-support-ive controls, respectively. The effect of PLGA on the adherence, proliferation and apoptosis of SCs were exam-ined in vitro in comparison with gelatin sponge and TCPS. Results: The micro-channels arrayed in parallel manners, and the pore sizes of the channels were uniform. No significant difference was found in the activity of Schwann cells cultured on PLEA and those on TCPS (P>0.05), and the DNA of PLGA scaffolds was not damaged. Conclusion: The 3-D scaffolds developed in this study have excellent structure and biocompatibility, which may be taken as a novel scaffold candidate for nerve-tissue engineering.

  8. Correlation between porous texture and cell seeding efficiency of gas foaming and microfluidic foaming scaffolds.

    Science.gov (United States)

    Costantini, Marco; Colosi, Cristina; Mozetic, Pamela; Jaroszewicz, Jakub; Tosato, Alessia; Rainer, Alberto; Trombetta, Marcella; Święszkowski, Wojciech; Dentini, Mariella; Barbetta, Andrea

    2016-05-01

    In the design of scaffolds for tissue engineering applications, morphological parameters such as pore size, shape, and interconnectivity, as well as transport properties, should always be tailored in view of their clinical application. In this work, we demonstrate that a regular and ordered porous texture is fundamental to achieve an even cell distribution within the scaffold under perfusion seeding. To prove our hypothesis, two sets of alginate scaffolds were fabricated using two different technological approaches of the same method: gas-in-liquid foam templating. In the first one, foam was obtained by insufflating argon in a solution of alginate and a surfactant under stirring. In the second one, foam was generated inside a flow-focusing microfluidic device under highly controlled and reproducible conditions. As a result, in the former case the derived scaffold (GF) was characterized by polydispersed pores and interconnects, while in the latter (μFL), the porous structure was highly regular both with respect to the spatial arrangement of pores and interconnects and their monodispersity. Cell seeding within perfusion bioreactors of the two scaffolds revealed that cell population inside μFL scaffolds was quantitatively higher than in GF. Furthermore, seeding efficiency data for μFL samples were characterized by a lower standard deviation, indicating higher reproducibility among replicates. Finally, these results were validated by simulation of local flow velocity (CFD) inside the scaffolds proving that μFL was around one order of magnitude more permeable than GF.

  9. Electrical stimulation of nerve cells using conductive nanofibrous scaffolds for nerve tissue engineering.

    Science.gov (United States)

    Ghasemi-Mobarakeh, Laleh; Prabhakaran, Molamma P; Morshed, Mohammad; Nasr-Esfahani, Mohammad Hossein; Ramakrishna, Seeram

    2009-11-01

    Fabrication of scaffolds with suitable chemical, mechanical, and electrical properties is critical for the success of nerve tissue engineering. Electrical stimulation was directly applied to electrospun conductive nanofibrous scaffolds to enhance the nerve regeneration process. In the present study, electrospun conductive nanofibers were prepared by mixing 10 and 15 wt% doped polyaniline (PANI) with poly (epsilon-caprolactone)/gelatin (PG) (70:30) solution (PANI/PG) by electrospinning. The fiber diameter, pore size, hydrophilicity, tensile properties, conductivity, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy spectra of nanofibers were determined, and the in vitro biodegradability of the different nanofibrous scaffolds was also evaluated. Nanofibrous scaffolds containing 15% PANI was found to exhibit the most balanced properties to meet all the required specifications for electrical stimulation for its enhanced conductivity and is used for in vitro culture and electrical stimulation of nerve stem cells. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and scanning electron microscopy results showed that conductive nanofibrous scaffolds are suitable substrates for the attachment and proliferation of nerve stem cells. Electrical stimulation through conductive nanofibrous PANI/PG scaffolds showed enhanced cell proliferation and neurite outgrowth compared to the PANI/PG scaffolds that were not subjected to electrical stimulation.

  10. Periodontal regeneration with stem cells-seeded collagen-hydroxyapatite scaffold.

    Science.gov (United States)

    Liu, Zeping; Yin, Xing; Ye, Qingsong; He, Wulin; Ge, Mengke; Zhou, Xiaofu; Hu, Jing; Zou, Shujuan

    2016-07-01

    Re-establishing compromised periodontium to its original structure, properties and function is demanding, but also challenging, for successful orthodontic treatment. In this study, the periodontal regeneration capability of collagen-hydroxyapatite scaffolds, seeded with bone marrow stem cells, was investigated in a canine labial alveolar bone defect model. Bone marrow stem cells were isolated, expanded and characterized. Porous collagen-hydroxyapatite scaffold and cross-linked collagen-hydroxyapatite scaffold were prepared. Attachment, migration, proliferation and morphology of bone marrow stem cells, co-cultured with porous collagen-hydroxyapatite or cross-linked collagen-hydroxyapatite, were evaluated in vitro. The periodontal regeneration capability of collagen-hydroxyapatite scaffold with or without bone marrow stem cells was tested in six beagle dogs, with each dog carrying one sham-operated site as healthy control, and three labial alveolar bone defects untreated to allow natural healing, treated with bone marrow stem cells - collagen-hydroxyapatite scaffold implant or collagen-hydroxyapatite scaffold implant, respectively. Animals were euthanized at 3 and 6 months (3 animals per group) after implantation and the resected maxillary and mandibular segments were examined using micro-computed tomography scan, H&E staining, Masson's staining and histometric evaluation. Bone marrow stem cells were successfully isolated and demonstrated self-renewal and multi-potency in vitro. The porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite had average pore sizes of 415 ± 20 µm and 203 ± 18 µm and porosity of 69 ± 0.5% and 50 ± 0.2%, respectively. The attachment, proliferation and migration of bone marrow stem cells were satisfactory on both porous collagen-hydroxyapatite and cross-linked collagen-hydroxyapatite scaffolds. Implantation of bone marrow stem cells - collagen-hydroxyapatite or collagen-hydroxyapatite scaffold in

  11. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior

    Science.gov (United States)

    Czisch, Christopher; Witek, Lukasz; Shi, Yang; Smay, Jim; Plant, Giles W.; Yang, Yunzhi

    2015-01-01

    Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin–3 (nt–3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts. PMID:26444999

  12. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Directory of Open Access Journals (Sweden)

    Lauren Sweet

    Full Text Available Numerous studies have demonstrated that Schwann cells (SCs play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP scaffolds arranged in 3D printed-lattice (P-β-TCP and randomly-porous, template-casted (N-β-TCP structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf, neutrophin-3 (nt-3, platelet-derived growth factor (pdgf-bb, and vascular endothelial growth factor (vegf-a were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  13. Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration.

    Science.gov (United States)

    Rathbone, C R; Guda, T; Singleton, B M; Oh, D S; Appleford, M R; Ong, J L; Wenke, J C

    2014-05-01

    Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration.

  14. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Science.gov (United States)

    Sweet, Lauren; Kang, Yunqing; Czisch, Christopher; Witek, Lukasz; Shi, Yang; Smay, Jim; Plant, Giles W; Yang, Yunzhi

    2015-01-01

    Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin-3 (nt-3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  15. A three-dimensional collagen scaffold cell culture system for screening anti-glioma therapeutics

    Science.gov (United States)

    Lv, Donglai; Yu, Shi-cang; Ping, Yi-fang; Wu, Haibo; Zhao, Xilong; Zhang, Huarong; Cui, Youhong; Chen, Bing; Zhang, Xia; Dai, Jianwu

    2016-01-01

    Three-dimensional (3D) culture, which can simulate in vivo microenvironments, has been increasingly used to study tumor cell biology. Since most preclinical anti-glioma drug tests still rely on conventional 2D cell culture, we established a collagen scaffold for 3D glioma cell culture. Glioma cells cultured on these 3D scaffolds showed greater degree of dedifferentiation and quiescence than cells in 2D culture. 3D-cultured cells also exhibited enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of O6-methylguanine DNA methyltransferase (MGMT). Importantly, tumor cells in 3D culture showed chemotherapy resistance patterns similar to those observed in glioma patients. Our results suggest that 3D collagen scaffolds are promising in vitro research platforms for screening new anti-glioma therapeutics. PMID:27486877

  16. Synthetic Polymer Scaffolds for Stem Cell Transplantation in Retinal Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Michael J. Young

    2011-05-01

    Full Text Available Age-related macular degeneration and retinitis pigmentosa are two leading causes of irreversible blindness characterized by photoreceptor loss. Cell transplantation may be one of the most promising approaches of retinal repair. However, several problems hinder the success of retinal regeneration, including cell delivery and survival, limited cell integration and incomplete cell differentiation. Recent studies show that polymer scaffolds can address these three problems. This article reviews the current literature on synthetic polymer scaffolds used for stem cell transplantation, especially retinal progenitor cells. The advantages and disadvantages of different polymer scaffolds, the role of different surface modifications on cell attachment and differentiation, and controlled drug delivery are discussed. The development of material and surface modification techniques is vital in making cell transplantation a clinical success.

  17. Enhanced PC12 cells proliferation with self-assembled S-layer proteins scaffolds.

    Science.gov (United States)

    Babolmorad, Ghazal; Emtiazi, Giti; Ghaedi, Kamran; Jodeiri, Mohamad

    2015-01-01

    Finding 3D biocompatible and biodegradable scaffold is important in tissue engineering which plays a critical role in transplanting methods. Several biomaterials, such as poly-L-lactide, poly(lactic-co-glycolic acid), poly(L-lactic acid)/poly(lactide-co-glycolide), alginate, collagen gel, and so on, have been applied as scaffold to culture cells in 3D environment. The most significant problem of the synthetic materials is lack of biocompatibility and bioactivity. Herein, self assemble S-layer proteins are used as a scaffold for PC12 cells culturing. For this purpose, S-layer protein was extracted from Bacillus coagulans HN68. Then, extracted S-layer was studied by SDS page and AFM. Using MTS test and Immunochemistry staining methods, the effect of self assembled S-layer scaffold on proliferation of PC12 cells was assayed. This study provides that S-layer could be an appropriate scaffold for PC12 cells culturing. Even though poly-L-ornithine is a common scaffold in PC12 cells culturing, the results show that (PLO)/S-layer is more protective.

  18. [Effect of polymeric scaffolds on attachment and growth of bone marrow mesenchymal stem cells].

    Science.gov (United States)

    Ren, Jie; Jia, Xiaozhen; Wang, Shuhong; Wu, Zhigang; Pan, Kefeng

    2005-12-01

    To investigate the effect of three kinds of polymeric scaffolds on attachment, proliferation and differentiation of bone marrow mesenchymal stem cells, the cells were different polymeric scaffolds of PLA-PEG, PLA, PLGA, respectively. The proliferation of cell was evaluated by cell count; the attachment and morphology of BMSCs were observed by SEM; and differentiation was detected by alkaline phosphatase activity, fluorescence, and RT-PCR methods. Results showed that the cells in PLGA group spread better among BMSCs adhered to the three polymeric scaffolds. The activity of ALP was detected after 3 days culture in these three groups. There were no significant differences between PLA-PEG and PLGA groups, but the activity of ALP was higher than PLA group. The gene expressions of osteocalicin and collagen I were also observed in the early culture time. Calcium nodes formation in these polymeric scaffolds were detected. BMSC spreading first, then overlapping growth and secretion of matrix around the bottom and surface of scaffolds were observed through SEM. In summary, PLA-PEG and PLGA are better polymeric scaffolds for the bone tissue engineering, compared with PLA.

  19. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  20. Plasma treatment for improving cell biocompatibility of a biodegradable polymer scaffold for vascular graft applications.

    Science.gov (United States)

    Valence, Sarra de; Tille, Jean-Christophe; Chaabane, Chiraz; Gurny, Robert; Bochaton-Piallat, Marie-Luce; Walpoth, Beat H; Möller, Michael

    2013-09-01

    Biodegradable synthetic scaffolds are being evaluated by many groups for the application of vascular tissue engineering. In addition to the choice of the material and the structure of the scaffold, tailoring the surface properties can have an important effect on promoting adequate tissue regeneration. The objective of this study was to evaluate the effect of an increased hydrophilicity of a polycaprolactone vascular graft by treatment with a cold air plasma. To this end, treated and untreated scaffolds were characterized, evaluated in vitro with smooth muscle cells, and implanted in vivo in the rat model for 3 weeks, both in the subcutaneous location and as an aortic replacement. The plasma treatment significantly increased the hydrophilicity of the scaffold, with complete wetting after a treatment of 60 sec, but did not change fiber morphology or mechanical properties. Smooth muscle cells cultured on plasma treated patches adopt a spread out morphology compared to a small, rounded morphology on untreated patches. Subcutaneous implantation revealed a low foreign body reaction for both types of scaffolds and a more extended and dense cellular infiltrate in the plasma treated scaffolds. In the vascular position, the plasma treatment induced a better cellularization of the graft wall, while it did not affect endothelialization rate or intimal hyperplasia. Plasma treatment is therefore an accessible tool to easily increase the biocompatibility of a scaffold and accelerate tissue regeneration without compromising mechanical strength, which are valuable advantages for vascular tissue engineering.

  1. Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

    Science.gov (United States)

    Rath, Subha N; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E; Kneser, Ulrich

    2012-10-01

    In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

  2. Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds

    Science.gov (United States)

    Brackmann, Christian; Esguerra, Maricris; Olausson, Daniel; Delbro, Dick; Krettek, Alexandra; Gatenholm, Paul; Enejder, Annika

    2011-02-01

    The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH2-groups at 2845 cm-1 permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.

  3. Graphene-augmented nanofiber scaffolds demonstrate new features in cells behaviour

    Science.gov (United States)

    Kazantseva, Jekaterina; Ivanov, Roman; Gasik, Michael; Neuman, Toomas; Hussainova, Irina

    2016-07-01

    Three-dimensional (3D) customized scaffolds capable to mimic a native extracellular matrix open new frontiers in cells manipulation and advanced therapy. The major challenge is in a proper substrate for in vitro models on engineered scaffolds, capable to modulate cells differentiation. Here for the first time we demonstrate novel design and functionality of the 3D porous scaffolds of aligned, self-assembled ceramic nanofibers of ultra-high anisotropy ratio (~107), augmented into graphene shells. This unique hybrid nano-network allows an exceptional combination of selective guidance stimuli of stem cells differentiation, immune reactions variations, and local immobilization of cancer cells, which was not available before. The scaffolds were shown to be able to direct human mesenchymal stem cells (important for stimulation of neuronal and muscle cells) preferential orientation, to suppress major inflammatory factors, and to localize cancer cells; all without additions of specific culture media. The selective downregulation of specific cytokines is anticipated as a new tool for understanding of human immune system and ways of treatment of associated diseases. The effects observed are self-regulated by cells only, without side effects, usually arising from use of external factors. New scaffolds may open new horizons for stem cells fate control such as towards axons and neurites regeneration (Alzheimer’s disease) as well as cancer therapy development.

  4. Compatibility of olfactory ensheathing cells with functionalized self-assembling peptide scaffold in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ling-ling; HUANG Lin-hong; ZHANG Zhen-xing; HAO Ding-jun; HE Bao-rong

    2013-01-01

    Background Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI).However,the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI.To improve the outcome,proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration.Methods In this study,we made a new peptide hydrogel scaffold named GRGDSPmx by mixing the pure RADA16 and designer peptide RADA16-GRGDSP solution,and we examined the molecular integration of the mixed nanofiber scaffolds using atomic force microscopy.In addition,we have studied the behavior of OECs in GRGDSPmx condition as well as on RADA16 scaffold by analyzing their phenotypes including cell proliferation,apoptosis,survival,and morphology.Results The experimental results showed that GRGDSPmx could be self-assembled to form a hydrogel.Inverted optical microscopic and scanning electron microscopic analyses showed that OECs are viable and they proliferate within the nanostructured environment of the scaffold.Thiazolyl blue (MTT) assay demonstrated that OEC proliferation rate was increased on GRGDSPmx scaffold compared with the pure RADA16 scaffold.In addition,OECs on GRGDSPmx scaffolds also showed less apoptosis and maintained the original spindle-shaped morphology.Calcein-AM/PI fluorescence staining revealed that OECs cultured on GRGDSPmx grew well and the viable cell count was 95%.Conclusion These results suggested that this new hydrogel scaffold provided an ideal substrate for OEC threedimensional culture and suggested its further application for SCI repair.

  5. The Effect of Aligned and Random Electrospun Fibrous Scaffolds on Rat Mesenchymal Stem Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Zahra Zonoubi

    2012-01-01

    Full Text Available Objective: The development of combining mesenchymal stem cells (MSCs with surface modified three-dimensional (3D biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL and unaltered PCL scaffolds were compared,and then p-PCL scaffolds were evaluated for MSC culture.Materials and Methods: Aligned and random PCL nanofibrus scaffolds were fabricatedby electrospining and their surface modified with O2 plasma treatment to enhanceMSC proliferation, adhesion and interaction. Chemical and mechanical characterizationswere carried out using scanning electron microscopy (SEM, water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance(ANOVA.Results: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl-2,5-DiphenyltetrazoliumBromide(MTT assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber.Conclusion: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.

  6. Immobilization of RGD Peptides onto Decellularized Valve Scaffolds to Promote Cell Adhesion

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD(arginine-glycine-aspartic acid) containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

  7. Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro

    Science.gov (United States)

    Calabrese, Giovanna; Forte, Stefano; Gulino, Rosario; Cefalì, Francesco; Figallo, Elisa; Salvatorelli, Lucia; Maniscalchi, Eugenia T.; Angelico, Giuseppe; Parenti, Rosalba; Gulisano, Massimo; Memeo, Lorenzo; Giuffrida, Raffaella

    2017-01-01

    Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could

  8. Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Puente, Pilar de la, E-mail: pilardelapuentegarcia@gmail.com [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain); Ludeña, Dolores [Pathology Service, University Hospital of Salamanca, P/San Vicente 58-182, 37007 Salamanca (Spain); López, Marta; Ramos, Jennifer; Iglesias, Javier [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain)

    2013-02-01

    Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.

  9. Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

    Directory of Open Access Journals (Sweden)

    Feng Yan

    2015-01-01

    Full Text Available In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold in vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hitosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial fibrillary acidic protein and a low level of expression of neuron-specific enolase were visible in BrdU-labeled bone marrow mesenchymal stem cells. These findings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold has a neuroprotective effect following ischemic stroke.

  10. Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies.

    Science.gov (United States)

    Stampella, A; Rizzitelli, G; Donati, F; Mazzarino, M; de la Torre, X; Botrè, F; Giardi, M F; Dentini, M; Barbetta, A; Massimi, M

    2015-12-25

    Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.

  11. Parameterizing the Transport Pathways for Cell Invasion in Complex Scaffold Architectures

    OpenAIRE

    Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G.; Best, Serena M.; Cameron, Ruth E.

    2016-01-01

    Interconnecting pathways through porous tissue engineering scaffolds play a vital role in determining nutrient supply, cell invasion, and tissue ingrowth. However, the global use of the term “interconnectivity” often fails to describe the transport characteristics of these pathways, giving no clear indication of their potential to support tissue synthesis. This article uses new experimental data to provide a critical analysis of reported methods for the description of scaffold transport pathw...

  12. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.

    Directory of Open Access Journals (Sweden)

    Fabrizio Gelain

    Full Text Available Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2. These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with beta-Tubulin(+, GFAP(+ and Nestin(+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.

  13. Thermoforming techniques for manufacturing porous scaffolds for application in 3D cell cultivation.

    Science.gov (United States)

    Borowiec, Justyna; Hampl, Jörg; Gebinoga, Michael; Elsarnagawy, Tarek; Elnakady, Yasser A; Fouad, Hassan; Almajhadi, Fahd; Fernekorn, Uta; Weise, Frank; Singh, Sukhdeep; Elsarnagawy, Dief; Schober, Andreas

    2015-04-01

    Within the scientific community, there is an increasing demand to apply advanced cell cultivation substrates with increased physiological functionalities for studying spatially defined cellular interactions. Porous polymeric scaffolds are utilized for mimicking an organ-like structure or engineering complex tissues and have become a key element for three-dimensional (3D) cell cultivation in the meantime. As a consequence, efficient 3D scaffold fabrication methods play an important role in modern biotechnology. Here, we present a novel thermoforming procedure for manufacturing porous 3D scaffolds from permeable materials. We address the issue of precise thermoforming of porous polymer foils by using multilayer polymer thermoforming technology. This technology offers a new method for structuring porous polymer foils that are otherwise available for non-porous polymers only. We successfully manufactured 3D scaffolds from solvent casted and phase separated polylactic acid (PLA) foils and investigated their biocompatibility and basic cellular performance. The HepG2 cell culture in PLA scaffold has shown enhanced albumin secretion rate in comparison to a previously reported polycarbonate based scaffold with similar geometry.

  14. TRANSPLANTATION OF CRYOPRESERVED FETAL LIVER CELLS SEEDED INTO MACROPOROUS ALGINATE-GELATIN SCAFFOLDS IN RATS WITH LIVER FAILURE

    Directory of Open Access Journals (Sweden)

    D. V. Grizay

    2015-01-01

    Full Text Available Aim. To study the therapeutic potential of cryopreserved fetal liver cells seeded into macroporous alginategelatin scaffolds after implantation to omentum of rats with hepatic failure.Materials and methods.Hepatic failure was simulated by administration of 2-acetyl aminofl uorene followed partial hepatectomy. Macroporous alginate-gelatin scaffolds, seeded with allogenic cryopreserved fetal liver cells (FLCs were implanted into rat omentum. To prevent from colonization of host cells scaffolds were coated with alginate gel shell. Serum transaminase activity, levels of albumin and bilirubin as markers of hepatic function were determined during 4 weeks after failure model formation and scaffold implantation. Morphology of liver and scaffolds after implantation were examined histologically. Results. Macroporous alginate-gelatin scaffolds after implantation to healthy rats were colonized by host cells. Additional formation of alginate gel shell around scaffolds prevented the colonization. Implantation of macroporous scaffolds seeded with cryopreserved rat FLCs and additionally coated with alginate gel shell into omentum of rats with hepatic failure resulted in signifi cant improvement of hepatospecifi c parameters of the blood serum and positive changes of liver morphology. The presence of cells with their extracellular matrix within the scaffolds was confi rmed after 4 weeks post implantation.Conclusion. The data above indicate that macroporous alginate-gelatin scaffolds coated with alginate gel shell are promising cell carriers for the development of bioengineered liver equivalents.

  15. Antitumor immunomodulatory activity of allogenic bone marrow cells on TiNi scaffold

    Science.gov (United States)

    Kokorev, O. V.; Hodorenko, V. N.; Cherdyntseva, N. V.; Gunther, V. E.

    2016-08-01

    The present study was undertaken to evaluate the feasibility of modulation of anti-tumor response by allogenic bone marrow cell transplantation into porous TiNi-based scaffold. Transplantation of bone marrow cells into porous TiNi-based scaffold leads to antitumor (35%) and antimetastatic (55%) effects. The lifetime of tumor-bearing animals and implanted allogenic bone marrow cells in incubator of TiNi increases up to 60%. The possible mechanisms of the effect of allogenic cells on tumor process are the stimulation of endogenous effectors of antitumor immunity.

  16. Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models.

    Science.gov (United States)

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena

    2014-01-01

    We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

  17. Cytocompatibility of brushite and monetite cell culture scaffolds made by three-dimensional powder printing.

    Science.gov (United States)

    Klammert, U; Reuther, T; Jahn, C; Kraski, B; Kübler, A C; Gbureck, U

    2009-02-01

    This study investigated the cytocompatibility of low-temperature direct 3-D printed calcium phosphate scaffolds in vitro. The fabrication of the scaffolds was performed with a commercial 3-D powder printing system. Diluted phosphoric acid was printed into tricalcium phosphate powder, leading to the formation of dicalcium phosphate dihydrate (brushite). Hydrothermal conversion of the brushite matrices led to the formation of dicalcium phosphate anhydrous (monetite). The biocompatibility was investigated using the osteoblastic cell line MC3T3-E1. Cell viability and the expression of alkaline phosphatase served as parameters. The culture medium was analyzed for pH value, concentration of free calcium and phosphate ions and osteocalcin. Both types of scaffolds showed a considerable increase of cell proliferation and viability; the monetite matrices were a little inferior compared with the brushite ones. The activity of alkaline phosphatase showed a similar pattern. Optical and electron microscopy revealed an obvious cell growth on the surface of both materials. Analysis of the culture medium showed minor alterations of pH value within the physiological range. The concentrations of free calcium and phosphate ions were obviously different among brushite and monetite cultures, due to their different solubility. The content of osteocalcin of the culture medium was reduced by the printed scaffolds due to adsorption. We conclude that the powder printed brushite and monetite matrices have a suitable biocompatibility for their use as cell culture scaffolds. Both materials enable osteoblastic cells in vitro to proliferate and differentiate due to the expression of typical osteoblastic markers.

  18. Differentiation of Wharton’s jelly mesenchymal stem cells into neurons in alginate scaffold

    Institute of Scientific and Technical Information of China (English)

    Seyed Mojtaba Hosseini; Attiyeh Vasaghi; Newsha Nakhlparvar; Reza Roshanravan; Tahereh Talaei-khozani; Zahra Razi

    2015-01-01

    Alginate scaffold has been considered as an appropriate biomaterial for promoting the differ-entiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton’s jelly mesenchymal stem cells (WJMSCs) and can pro-mote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 µM retinoic acid and 20 ng/mL basic ifbroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunolfuorescence staining was performed for detectingβ-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker).β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

  19. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

    Science.gov (United States)

    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  20. Valvular interstitial cell seeded poly(glycerol sebacate) scaffolds: toward a biomimetic in vitro model for heart valve tissue engineering.

    Science.gov (United States)

    Masoumi, Nafiseh; Johnson, Katherine L; Howell, M Christian; Engelmayr, George C

    2013-04-01

    Tissue engineered replacement heart valves may be capable of overcoming the lack of growth potential intrinsic to current non-viable prosthetics, and thus could potentially serve as permanent replacements in the surgical repair of pediatric valvular lesions. However, the evaluation of candidate combinations of cells and scaffolds lacks a biomimetic in vitro model with broadly tunable, anisotropic and elastomeric structural-mechanical properties. Toward establishing such an in vitro model, in the current study, porcine aortic and pulmonary valvular interstitial cells (i.e. biomimetic cells) were cultivated on anisotropic, micromolded poly(glycerol sebacate) scaffolds (i.e. biomimetic scaffolds). Following 14 and 28 days of static culture, cell-seeded scaffolds and unseeded controls were assessed for their mechanical properties, and cell-seeded scaffolds were further characterized by confocal fluorescence and scanning electron microscopy, and by collagen and DNA assays. Poly(glycerol sebacate) micromolding yielded scaffolds with anisotropic stiffnesses resembling those of native valvular tissues in the low stress-strain ranges characteristic of physiologic valvular function. Scaffold anisotropy was largely retained upon cultivation with valvular interstitial cells; while the mechanical properties of unseeded scaffolds progressively diminished, cell-seeded scaffolds either retained or exceeded initial mechanical properties. Retention of mechanical properties in cell-seeded scaffolds paralleled the accretion of collagen, which increased significantly from 14 to 28 days. This study demonstrates that valvular interstitial cells can be cultivated on anisotropic poly(glycerol sebacate) scaffolds to yield biomimetic in vitro models with which clinically relevant cells and future scaffold designs can be evaluated.

  1. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    Science.gov (United States)

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

  2. Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials

    Directory of Open Access Journals (Sweden)

    A Gigante

    2009-08-01

    Full Text Available Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.

  3. Fabrication of nanofibrous scaffold using a PLA and hagfish thread keratin composite; its effect on cell adherence, growth, and osteoblast differentiation.

    Science.gov (United States)

    Kim, Beom-Su; Park, Ko Eun; Park, Won Ho; Lee, Jun

    2013-08-01

    Electrospinning is a useful method for the production of nanofibrous scaffolds in the field of tissue engineering. Keratin has been used as a biomaterial for electrospinning and can be used in a variety of biomedical applications because it is a natural protein, giving it the ability to improve cell affinity of scaffolds. In this study, keratin was extracted from hagfish slime thread (H-keratin) and blended with polylactic acid (PLA) polymer solution to construct a nanofibrous scaffold. Wool keratin (W-keratin) was used as a control for the comparison of morphological, physical, and biological properties. The results of Fourier transform infrared spectroscopy showed the presence of both W-keratin and H-keratin in the electrospun PLA/keratin. Observations with a scanning electron microscope revealed that PLA, PLA/W-keratin, and PLA/H-keratin had similar average diameters (~800 nm). Cell attachment experiments showed that MG-63 cells adhered more rapidly and spread better onto PLA/H-keratin than onto the pure PLA or PLA/W-keratin. Cell proliferation assay, DNA content, live/dead, and alkaline phosphatase activity assays showed that PLA/H-keratin scaffolds could accelerate the viability, proliferation, and osteogenesis of MG-63 cells relative to pure PLA or PLA/W-keratin nanofibrous scaffolds. These findings suggest that H-keratin can improve cellular attraction and has great potential to be used as a biomaterial in bone tissue engineering.

  4. Effect of different calcium phosphate scaffold ratios on odontogenic differentiation of human dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    AbdulQader, Sarah Talib [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Department of Pedodontic and Preventive Dentistry, College of Dentistry, University of Baghdad, Baghdad (Iraq); Kannan, Thirumulu Ponnuraj, E-mail: kannan@usm.my [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Rahman, Ismail Ab [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Ismail, Hanafi [School of Materials and Minerals Resource Engineering, Universiti Sains Malaysia, 14300 Penang (Malaysia); Mahmood, Zuliani [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2015-04-01

    Calcium phosphate (CaP) scaffolds have been widely and successfully used with osteoblast cells for bone tissue regeneration. However, it is necessary to investigate the effects of these scaffolds on odontoblast cells' proliferation and differentiation for dentin tissue regeneration. In this study, three different hydroxyapatite (HA) to beta tricalcium phosphate (β-TCP) ratios of biphasic calcium phosphate (BCP) scaffolds, BCP20, BCP50, and BCP80, with a mean pore size of 300 μm and 65% porosity were prepared from phosphoric acid (H{sub 2}PO{sub 4}) and calcium carbonate (CaCO{sub 3}) sintered at 1000 °C for 2 h. The extracts of these scaffolds were assessed with regard to cell viability and differentiation of odontoblasts. The high alkalinity, more calcium, and phosphate ions released that were exhibited by BCP20 decreased the viability of human dental pulp cells (HDPCs) as compared to BCP50 and BCP80. However, the cells cultured with BCP20 extract expressed high alkaline phosphatase activity and high expression level of bone sialoprotein (BSP), dental matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) genes as compared to that cultured with BCP50 and BCP80 extracts. The results highlighted the effect of different scaffold ratios on the cell microenvironment and demonstrated that BCP20 scaffold can support HDPC differentiation for dentin tissue regeneration. - Highlights: • BCPs of different HA/β-TCP ratios influence cell microenvironment. • BCP20 decreases cell viability of HDPCs as compared to BCP50 and BCP80. • HDPCs cultured with BCP20 express highest ALP activity. • HDPCs cultured with BCP20 up-regulate BSP, DMP-1 and DSPP gene expressions. • BCP20 can support HDPC differentiation for dentin tissue regeneration.

  5. "Common synthetic scaffolds" in the synthesis of structurally diverse natural products.

    Science.gov (United States)

    Anagnostaki, Elissavet E; Zografos, Alexandros L

    2012-09-01

    Selected natural products have long been considered as desirable targets for total synthesis due to their unique biological properties and their challenging structural complexity. Laboratory synthesis of natural compounds usually relies on target-oriented synthesis, involving the production, isolation and purification of successive intermediates, requiring multiple steps to arrive to the final product. A far more economical approach using common synthetic scaffolds that can be readily transformed through biomimetic-like pathways to a range of structurally diverse natural products has been evolved in the last decade, leading synthesis to new directions. This tutorial review critically presents the hallmarks in this field.

  6. Biocompatibility Properties of Polyamide 6/ PCL Blends Composite Textile Scaffold using EA.hy926 Human Endothelial Cells.

    Science.gov (United States)

    Abdal-Hay, Abdalla; Abdelrazek Khalil, Khalil; Al-Jassir, Fawzi F; Gamal-Eldeen, Amira

    2017-02-27

    Enhancing the cytocompatibility profiles including cell attachment, growth and viability of designed synthetic scaffolds have a pivotal role in tissue engineering applications. Polymer blending is one of the most effective methods for providing new desirable biomaterials for tissue scaffolds. This article reports a novel polyamide 6/ poly(Ɛ-caprolactone) (PA6/PCL) blends solution by varying the concentrations ratios of PA6 and PCL which was fabricated to create composite fibrous tissue scaffolds. Highly porous blends fibrous scaffold has been fabricated and their suitability as cell-support for EA.hy926 human endothelial cells has been studied. Our results demonstrated that the unique nanoscale morphological properties and tune porosity of the blends scaffold were controlled. We found that these properties are mainly depending on the PA6/PCL blending viscosity value, and the viscosity of the blending solution has an intense effect on the properties of the blends scaffold. The influence of the scaffolds extraction fluids and the scaffold direct contact of both of the metabolic viability and the DNA integrity of EA.hy926 endothelial cells as well as the cell/ scaffold interaction analysis by Scanning Electron Microscope, after different co-culturing intervals, demonstrated that PA6/PCL blend scaffolds showed different behavior. Blend scaffolds of PA6/PCL of 90:10 ratio proved to be excellent endothelial cell carrier, which provided a good cell morphology, DNA integrity and viability, induced DNA synthesis/replication, and enhanced cell proliferation, attachment, and invasion. These results indicate that blends of PA6/PCL composite fibers is a promising 3D substitute for the next generation of synthetic tissue scaffold that could soon find clinical applications.

  7. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    Science.gov (United States)

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  8. Nanoparticulate mineralized collagen scaffolds induce in vivo bone regeneration independent of progenitor cell loading or exogenous growth factor stimulation.

    Science.gov (United States)

    Ren, Xiaoyan; Tu, Victor; Bischoff, David; Weisgerber, Daniel W; Lewis, Michael S; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2016-05-01

    Current strategies for skeletal regeneration often require co-delivery of scaffold technologies, growth factors, and cellular material. However, isolation and expansion of stem cells can be time consuming, costly, and requires an additional procedure for harvest. Further, the introduction of supraphysiologic doses of growth factors may result in untoward clinical side effects, warranting pursuit of alternative methods for stimulating osteogenesis. In this work, we describe a nanoparticulate mineralized collagen glycosaminoglycan scaffold that induces healing of critical-sized rabbit cranial defects without addition of expanded stem cells or exogenous growth factors. We demonstrate that the mechanism of osteogenic induction corresponds to an increase in canonical BMP receptor signalling secondary to autogenous production of BMP-2 and -9 early and BMP-4 later during differentiation. Thus, nanoparticulate mineralized collagen glycosaminoglycan scaffolds may provide a novel growth factor-free and ex vivo progenitor cell culture-free implantable method for bone regeneration.

  9. Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

    Science.gov (United States)

    Soman, Pranav; Tobe, Brian T D; Lee, Jin Woo; Winquist, Alicia A M; Singec, Ilyas; Vecchio, Kenneth S; Snyder, Evan Y; Chen, Shaochen

    2012-10-01

    Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

  10. Mechanical modulation of nascent stem cell lineage commitment in tissue engineering scaffolds.

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L

    2013-07-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to stem cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to "map the mechanome", defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates.

  11. Mechanical Modulation of Nascent Stem Cell Lineage Commitment in Tissue Engineering Scaffolds

    Science.gov (United States)

    Song, Min Jae; Dean, David; Tate, Melissa L. Knothe

    2013-01-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to “map the mechanome”, defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates. PMID:23660249

  12. Alginate based scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Valente, J.F.A.; Valente, T.A.M. [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Faculdade de Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal); Alves, P.; Ferreira, P. [CIEPQPF, Departamento de Engenharia Quimica, Universidade de Coimbra, Polo II, Pinhal de Marrocos, 3030-290 Coimbra (Portugal); Silva, A. [Centro de Ciencia e Tecnologia Aeroespaciais, Universidade da Beira Interior, Covilha (Portugal); Correia, I.J., E-mail: icorreia@ubi.pt [CICS-UBI - Centro de Investigacao em Ciencias da Saude, Faculdade de Ciencias da Saude, Universidade da Beira Interior, Covilha (Portugal)

    2012-12-01

    The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine. The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence. The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays. - Highlights: Black-Right-Pointing-Pointer Design and production of scaffolds for bone tissue regeneration. Black-Right-Pointing-Pointer Microparticle and microfiber alginate scaffolds were produced through a particle aggregation technique; Black-Right-Pointing-Pointer Scaffolds' mechanically and biologically properties were characterized through in vitro studies;.

  13. Investigation the Porous Collagen-Chitosan /Glycosaminoglycans for Corneal Cell Culture as Tissue Engineering Scaffold

    Institute of Scientific and Technical Information of China (English)

    LI Qin-Hua; CHEN Jian-Su

    2005-01-01

    The objective of this study was to produce the porous collagen-chitosan/Glycosanminglycans (GAG) for corneal ceil-seed implant as a three-dimensional tissue engineering scaffold to improve the regeneration corneas. The effect of various content of glycerol as form porous agent to collagen-chitosan/GAG preserved a porous dimensional structure was investigated. The heat-drying was used to prepare porous collagen-chitosan /GAG scaffold. The pore morphology of collagenchitosan/GAG was controlled by changing the concentration of glycerol solution and drying methods. The porous structure morphology was observed by SEM. The diameter of the pores form 10 to 50 μm. The highly porous scaffold had interconnecting pores. The corneal cell morphology was observed under the light microscope. These results suggest that collagen-chitosan/GAG showed that corneal cell have formed confluent layers and resemble the surface of normal corneal cell surface.

  14. Fibrin Gel as an Injectable Biodegradable Scaffold and Cell Carrier for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Yuting Li

    2015-01-01

    Full Text Available Due to the increasing needs for organ transplantation and a universal shortage of donated tissues, tissue engineering emerges as a useful approach to engineer functional tissues. Although different synthetic materials have been used to fabricate tissue engineering scaffolds, they have many limitations such as the biocompatibility concerns, the inability to support cell attachment, and undesirable degradation rate. Fibrin gel, a biopolymeric material, provides numerous advantages over synthetic materials in functioning as a tissue engineering scaffold and a cell carrier. Fibrin gel exhibits excellent biocompatibility, promotes cell attachment, and can degrade in a controllable manner. Additionally, fibrin gel mimics the natural blood-clotting process and self-assembles into a polymer network. The ability for fibrin to cure in situ has been exploited to develop injectable scaffolds for the repair of damaged cardiac and cartilage tissues. Additionally, fibrin gel has been utilized as a cell carrier to protect cells from the forces during the application and cell delivery processes while enhancing the cell viability and tissue regeneration. Here, we review the recent advancement in developing fibrin-based biomaterials for the development of injectable tissue engineering scaffold and cell carriers.

  15. Pulmonary heart valve replacement using stabilized acellular xenogeneic scaffolds; effects of seeding with autologous stem cells

    Directory of Open Access Journals (Sweden)

    Harpa Marius Mihai

    2015-12-01

    Full Text Available Background: We hypothesized that an ideal heart valve replacement would be acellular valve root scaffolds seeded with autologous stem cells. To test this hypothesis, we prepared porcine acellular pulmonary valves, seeded them with autologous adipose derived stem cells (ADSCs and implanted them in sheep and compared them to acellular valves.

  16. Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats.

    Science.gov (United States)

    Goyal, Ritu; Guvendiren, Murat; Freeman, Onyi; Mao, Yong; Kohn, Joachim

    2017-01-11

    The design of composite tissue scaffolds containing an extracellular matrix (ECM) and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000), medium (E0500), and fast (E1000) degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw) retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days) was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days) did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days) allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds.

  17. Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats

    Directory of Open Access Journals (Sweden)

    Ritu Goyal

    2017-01-01

    Full Text Available The design of composite tissue scaffolds containing an extracellular matrix (ECM and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000, medium (E0500, and fast (E1000 degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds.

  18. Fiber diameter and seeding density influence chondrogenic differentiation of mesenchymal stem cells seeded on electrospun poly(ε-caprolactone) scaffolds.

    Science.gov (United States)

    Bean, Allison C; Tuan, Rocky S

    2015-01-29

    Chondrogenic differentiation of mesenchymal stem cells is strongly influenced by the surrounding chemical and structural milieu. Since the majority of the native cartilage extracellular matrix is composed of nanofibrous collagen fibrils, much of recent cartilage tissue engineering research has focused on developing and utilizing scaffolds with similar nanoscale architecture. However, current literature lacks consensus regarding the ideal fiber diameter, with differences in culture conditions making it difficult to compare between studies. Here, we aimed to develop a more thorough understanding of how cell-cell and cell-biomaterial interactions drive in vitro chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs). Electrospun poly(ε-caprolactone) microfibers (4.3  ±  0.8 µm diameter, 90 μm(2) pore size) and nanofibers (440  ±  20 nm diameter, 1.2 μm(2) pore size) were seeded with MSCs at initial densities ranging from 1  ×  10(5) to 4  ×  10(6) cells cm(-3)-scaffold and cultured under transforming growth factor-β (TGF-β) induced chondrogenic conditions for 3 or 6 weeks. Chondrogenic gene expression, cellular proliferation, as well as sulfated glycosaminoglycan and collagen production were enhanced on microfiber in comparison to nanofiber scaffolds, with high initial seeding densities being required for significant chondrogenic differentiation and extracellular matrix deposition. Both cell-cell and cell-material interactions appear to play important roles in chondrogenic differentiation of MSCs in vitro and consideration of several variables simultaneously is essential for understanding cell behavior in order to develop an optimal tissue engineering strategy.

  19. Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

    Energy Technology Data Exchange (ETDEWEB)

    Bernemann, Inga, E-mail: bernemann@imp.uni-hannover.de [Institute for Multiphase Processes, Leibniz Universitaet Hannover, Hannover (Germany); Mueller, Thomas; Blasczyk, Rainer [Institute for Transfusion Medicine, Hannover Medical School, Hannover (Germany); Glasmacher, Birgit; Hofmann, Nicola [Institute for Multiphase Processes, Leibniz Universitaet Hannover, Hannover (Germany)

    2011-07-29

    Highlights: {yields} Marmoset bone marrow-derived MSCs differentiate in suspension into adipogenic, osteogenic and chondrogenic lineages. {yields} Marmoset MSCs integrate in collagen type I scaffolds and differentiate excellently into adipogenic cells. {yields} Common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. -- Abstract: In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 {mu}m were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential

  20. Textile cell-free scaffolds for in situ tissue engineering applications.

    Science.gov (United States)

    Aibibu, Dilbar; Hild, Martin; Wöltje, Michael; Cherif, Chokri

    2016-03-01

    In this article, the benefits offered by micro-fibrous scaffold architectures fabricated by textile manufacturing techniques are discussed: How can established and novel fiber-processing techniques be exploited in order to generate templates matching the demands of the target cell niche? The problems related to the development of biomaterial fibers (especially from nature-derived materials) ready for textile manufacturing are addressed. Attention is also paid on how biological cues may be incorporated into micro-fibrous scaffold architectures by hybrid manufacturing approaches (e.g. nanofiber or hydrogel functionalization). After a critical review of exemplary recent research works on cell-free fiber based scaffolds for in situ TE, including clinical studies, we conclude that in order to make use of the whole range of favors which may be provided by engineered fibrous scaffold systems, there are four main issues which need to be addressed: (1) Logical combination of manufacturing techniques and materials. (2) Biomaterial fiber development. (3) Adaption of textile manufacturing techniques to the demands of scaffolds for regenerative medicine. (4) Incorporation of biological cues (e.g. stem cell homing factors).

  1. Variation of mechanical behavior of β-TCP/collagen two phase composite scaffold with mesenchymal stem cell in vitro.

    Science.gov (United States)

    Arahira, Takaaki; Todo, Mitsugu

    2016-08-01

    The primary aim of this study is to characterize the variational behavior of the compressive mechanical property of bioceramic-based scaffolds using stem cells during the cell culture period. β-Tricalcium phosphate (TCP)/collagen two phase composites and β-TCP scaffolds were fabricated using the polyurethane template technique and a subsequent freeze-drying method. Rat bone-marrow mesenchymal stem cells (rMSCs) were then cultured in these scaffolds for up to 28 days. Compression tests of the scaffolds with rMSCs were periodically conducted. Biological properties, such as the cell number, alkaline phosphatase (ALP) activity, and gene expressions of osteogenesis, were evaluated. The microstructural change due to cell growth and the formation of extracellular matrices was examined using a field-emission scanning electron microscope. The compressive property was then correlated with the biological properties and microstructures to understand the mechanism of the variational behavior of the macroscopic mechanical property. The porous collagen structure in the β-TCP scaffold effectively improved the structural stability of the composite scaffold, whereas the β-TCP scaffold exhibited structural instability with the collapse of the porous structure when immersed in a culture medium. The β-TCP/collagen composite scaffold exhibited higher ALP activity and more active generation of osteoblastic markers than the β-TCP scaffold.

  2. Gel-cast glass-ceramic tissue scaffolds of controlled architecture produced via stereolithography of moulds.

    Science.gov (United States)

    Chopra, K; Mummery, P M; Derby, B; Gough, J E

    2012-12-01

    Two glass-ceramic scaffolds with a simple cubic structure of 500 µm square ligaments and square channels of width 400 or 600 µm have been fabricated by gel-casting into moulds produced by stereolithography, followed by mould removal, polymer burnout and sintering. The scaffolds have crushing strengths of 41 ± 14 and 17 ± 5 Mpa, respectively. Using a method of assembling discrete slices of scaffold, we are able to study cell behaviour within a scaffold by disassembly. Both scaffold structures were seeded with primary human osteoblasts and these penetrate, adhere, spread and proliferate on the scaffold structure. The larger channel diameter scaffold shows a greater cell population (despite its smaller surface area) and more pronounced production of ECM components (collagen and mineralization) with increased time in culture. Studies of sectioned scaffolds show that cell density and ECM production decrease with depth and that the difference between the two scaffold architectures is maintained.

  3. Design and characterization of core-shell mPEG-PLGA composite microparticles for development of cell-scaffold constructs

    DEFF Research Database (Denmark)

    Wen, Yanhong; Gallego, Monica Ramos; Nielsen, Lene Feldskov

    2013-01-01

    Appropriate scaffolds capable of providing suitable biological and structural guidance are of great importance to generate cell-scaffold constructs for cell-based tissue engineering. The aim of the present study was to develop composite microparticles with a structure to provide functionality as ...

  4. Tuning the Mechanical Properties of Poly(Ethylene Glycol) Microgel-Based Scaffolds to Increase 3D Schwann Cell Proliferation.

    Science.gov (United States)

    Zhou, Wenda; Stukel, Jessica M; Cebull, Hannah L; Willits, Rebecca Kuntz

    2016-04-01

    2D in vitro studies have demonstrated that Schwann cells prefer scaffolds with mechanical modulus approximately 10× higher than the modulus preferred by nerves, limiting the ability of many scaffolds to promote both neuron extension and Schwann cell proliferation. Therefore, the goals of this work are to develop and characterize microgel-based scaffolds that are tuned over the stiffness range relevant to neural tissue engineering and investigate Schwann cell morphology, viability, and proliferation within 3D scaffolds. Using thiol-ene reaction, microgels with surface thiols are produced and crosslinked into hydrogels using a multiarm vinylsulfone (VS). By varying the concentration of VS, scaffold stiffness ranges from 0.13 to 0.76 kPa. Cell morphology in all groups demonstrates that cells are able to spread and interact with the scaffold through day 5. Although the viability in all groups is high, proliferation of Schwann cells within the scaffold of G* = 0.53 kPa is significantly higher than other groups. This result is ≈ 5× lower than previously reported optimal stiffnesses on 2D surfaces, demonstrating the need for correlation of 3D cell response to mechanical modulus. As proliferation is the first step in Schwann cell integration into peripheral nerve conduits, these scaffolds demonstrate that the stiffness is a critical parameter to optimizing the regenerative process.

  5. Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes.

    Science.gov (United States)

    Bjerre, Lea; Bünger, Cody; Baatrup, Anette; Kassem, Moustapha; Mygind, Tina

    2011-06-01

    Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final number of cells was higher in 500 μm constructs as compared with 200 μm constructs. Alkaline phosphatase enzyme activity assays and real time RT-PCR on seven osteogenic markers showed that differentiation occurred primarily and earlier in statically cultured constructs with 200 μm pores compared with 500 μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology of scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation

  6. Laser 3D printing with sub-microscale resolution of porous elastomeric scaffolds for supporting human bone stem cells.

    Science.gov (United States)

    Petrochenko, Peter E; Torgersen, Jan; Gruber, Peter; Hicks, Lucas A; Zheng, Jiwen; Kumar, Girish; Narayan, Roger J; Goering, Peter L; Liska, Robert; Stampfl, Jürgen; Ovsianikov, Aleksandr

    2015-04-01

    A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

  7. Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Wu, Pai-Shuen [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Ko, Chih-Sheng [PhytoHealth Corporation, Maywufa Biopharma Group, Taipei, Taiwan (China); Huang, Te-Yang [Mackay Memorial Hospital, Taipei, Taiwan (China)

    2014-08-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering • Strontium phosphate can enhance the osteogenic differentiation of SHEDs • 3D scaffold under dynamic culture with optimal fluid rate enhance cellular differentiation.

  8. In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds

    Directory of Open Access Journals (Sweden)

    Hyeon Joo Kim

    2012-12-01

    Full Text Available Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell–silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.

  9. Effects of Multiwalled Carbon Nanotube Reinforced Collagen Scaffolds on the Osteogenic Differentiation of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Rena Baktur

    2013-01-01

    Full Text Available With recent advances in nanotechnology, carbon nanotubes (CNTs have been extensively studied as substrates for cell culture, drug delivery systems, and medical implant materials. However, surprisingly little is known about the effect of CNTs on collective cellular processes (e.g., adhesion, proliferation, and differentiation. This leads to the need for quantitative characterization of the proliferation, differentiation, and mineralization of mesenchymal stem cells (MSCs on multiwalled CNT-s (MWCNTs- collagen scaffolds. In here, a set of MWCNTs-collagen scaffolds where three different types of MWCNTs are, respectively, entrapped in reconstituted type I collagen at four different concentrations less than 100 ppm are prepared; the MSC differentiation thereon is investigated by monitoring the transcription factor RunX2 (RunX, transforming growth factor β (TGF-β, alkaline phosphatase (AP, osteocalcin, and mineralized nodules of extracellular matrix (ECM. In short, the MWCNT-collagen scaffolds induced significant increases in AP activity and ECM mineralization due to the increased stiffness and strength of the scaffold by entrapping MWCNTs. This offers a potential for controlling MSC differentiation using MWCNT-collagen scaffolds.

  10. Protein Corona Influences Cell-Biomaterial Interactions in Nanostructured Tissue Engineering Scaffolds.

    Science.gov (United States)

    Serpooshan, Vahid; Mahmoudi, Morteza; Zhao, Mingming; Wei, Ke; Sivanesan, Senthilkumar; Motamedchaboki, Khatereh; Malkovskiy, Andrey V; Gladstone, Andrew B; Cohen, Jeffrey E; Yang, Phillip C; Rajadas, Jayakumar; Bernstein, Daniel; Woo, Y Joseph; Ruiz-Lozano, Pilar

    2015-07-22

    Biomaterials are extensively used to restore damaged tissues, in the forms of implants (e.g. tissue engineered scaffolds) or biomedical devices (e.g. pacemakers). Once in contact with the physiological environment, nanostructured biomaterials undergo modifications as a result of endogenous proteins binding to their surface. The formation of this macromolecular coating complex, known as 'protein corona', onto the surface of nanoparticles and its effect on cell-particle interactions are currently under intense investigation. In striking contrast, protein corona constructs within nanostructured porous tissue engineering scaffolds remain poorly characterized. As organismal systems are highly dynamic, it is conceivable that the formation of distinct protein corona on implanted scaffolds might itself modulate cell-extracellular matrix interactions. Here, we report that corona complexes formed onto the fibrils of engineered collagen scaffolds display specific, distinct, and reproducible compositions that are a signature of the tissue microenvironment as well as being indicative of the subject's health condition. Protein corona formed on collagen matrices modulated cellular secretome in a context-specific manner ex-vivo, demonstrating their role in regulating scaffold-cellular interactions. Together, these findings underscore the importance of custom-designing personalized nanostructured biomaterials, according to the biological milieu and disease state. We propose the use of protein corona as in situ biosensor of temporal and local biomarkers.

  11. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  12. 3D collagen scaffolds coated with multiwalled carbon nanotubes: initial cell attachment to internal surface.

    Science.gov (United States)

    Hirata, Eri; Uo, Motohiro; Nodasaka, Yoshinobu; Takita, Hiroko; Ushijima, Natsumi; Akasaka, Tsukasa; Watari, Fumio; Yokoyama, Atsuro

    2010-05-01

    The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

  13. Semiotic scaffolding

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2015-01-01

    Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings...... implies that genes do not control the life of organisms, they merely scaffold it. The nature-nurture dynamics is thus far more complex and open than is often claimed. Contrary to physically based interactions, semiotic interactions do not depend on any direct causal connection between the sign vehicle...... semiotic scaffolding is not, of course, exclusive for phylogenetic and ontogenetic development, it is also an important dynamical element in cultural evolution....

  14. Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

    Directory of Open Access Journals (Sweden)

    María D. Cuenca-López

    2014-12-01

    Full Text Available The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft for posterolateral lumbar fusion (PLF in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM mesenchymal stem cells (MSCs have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group, hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct. During the last three days of culture, dexamethasone (dex and beta-glycerophosphate (β-GP were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5. Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT, histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70% than for mineral scaffold alone (22% and hybrid constructs (35%. The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft. Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored

  15. Evaluation of posterolateral lumbar fusion in sheep using mineral scaffolds seeded with cultured bone marrow cells.

    Science.gov (United States)

    Cuenca-López, María D; Andrades, José A; Gómez, Santiago; Zamora-Navas, Plácido; Guerado, Enrique; Rubio, Nuria; Blanco, Jerónimo; Becerra, José

    2014-12-16

    The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (β-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4-L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by

  16. Characterization of novel akermanite:poly-ϵ-caprolactone scaffolds for human adipose-derived stem cells bone tissue engineering.

    Science.gov (United States)

    Zanetti, A S; McCandless, G T; Chan, J Y; Gimble, J M; Hayes, D J

    2015-04-01

    In this study, three different akermanite:poly-ϵ-caprolactone (PCL) composite scaffolds (wt%: 75:25, 50:50, 25:75) were characterized in terms of structure, compression strength, degradation rate and in vitro biocompatibility to human adipose-derived stem cells (hASC). Pure ceramic scaffolds [CellCeram™, custom-made, 40:60 wt%; β-tricalcium phosphate (β-TCP):hydroxyapatite (HA); and akermanite] and PCL scaffolds served as experimental controls. Compared to ceramic scaffolds, the authors hypothesized that optimal akermanite:PCL composites would have improved compression strength and comparable biocompatibility to hASC. Electron microscopy analysis revealed that PCL-containing scaffolds had the highest porosity but CellCeram™ had the greatest pore size. In general, compression strength in PCL-containing scaffolds was greater than in ceramic scaffolds. PCL-containing scaffolds were also more stable in culture than ceramic scaffolds. Nonetheless, mass losses after 21 days were observed in all scaffold types. Reduced hASC metabolic activity and increased cell detachment were observed after acute exposure to akermanite:PCL extracts (wt%: 75:25, 50:50). Among the PCL-containing scaffolds, hASC cultured for 21 days on akermanite:PCL (wt%: 75:25) discs displayed the highest viability, increased expression of osteogenic markers (alkaline phosphatase and osteocalcin) and lowest IL-6 expression. Together, the results indicate that akermanite:PCL composites may have appropriate mechanical and biocompatibility properties for use as bone tissue scaffolds.

  17. Microfabrication of chip-sized scaffolds for three-dimensional cell cultivation.

    Science.gov (United States)

    Giselbrecht, Stefan; Gottwald, Eric; Truckenmueller, Roman; Trautmann, Christina; Welle, Alexander; Guber, Andreas; Saile, Volker; Gietzelt, Thomas; Weibezahn, Karl-Friedrich

    2008-05-12

    Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites. Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2 So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers. In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion

  18. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Wu XN

    2014-08-01

    Full Text Available Xiaonan Wu,1 Leiying Miao,2,# Yingfang Yao,3 Wenlei Wu,1 Yu Liu,1 Xiaofeng Chen,1 Weibin Sun1,# 1Department of Periodontology, Hospital of Stomatology, Medical School of Nanjing University, Nanjing, People’s Republic of China; 2Department of Cariology and Endodontics, Hospital of Stomatology, Medical School of Nanjing University, Nanjing, People’s Republic of China; 3Eco-materials and Renewable Energy Research Center, Department of Materials Science and Engineering, National Laboratory of Solid State Microstructures, Nanjing University, Nanjing, People’s Republic of China #These authors contributed equally to this work Abstract: Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(є-caprolactone (COL/PCL and type I collagen/poly(є-caprolactone/nanoscale hydroxyapatite (COL/PCL/nHA with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then

  19. Reconstruction of beagle hemi-mandibular defects with allogenic mandibular scaffolds and autologous mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    ChangKui Liu

    Full Text Available Massive bone allografts are frequently used in orthopedic reconstructive surgery, but carry a high failure rate of approximately 25%. We tested whether treatment of graft with mesenchymal stem cells (MSCs can increase the integration of massive allografts (hemi-mandible in a large animal model.Thirty beagle dogs received surgical left-sided hemi-mandibular defects, and then divided into two equal groups. Bony defects of the control group were reconstructed using allografts only. Those of the experimental group were reconstructed using allogenic mandibular scaffold-loaded autologous MSCs. Beagles from each group were killed at 4 (n = 4, 12 (n = 4, 24 (n = 4 or 48 weeks (n = 3 postoperatively. CT and micro-CT scans, histological analyses and the bone mineral density (BMD of transplants were used to evaluate defect reconstruction outcomes.Gross and CT examinations showed that the autologous bone grafts had healed in both groups. At 48 weeks, the allogenic mandibular scaffolds of the experimental group had been completely replaced by new bone, which has a smaller surface area to that of the original allogenic scaffold, whereas the scaffold in control dogs remained the same size as the original allogenic scaffold throughout. At 12 weeks, the BMD of the experimental group was significantly higher than the control group (p<0.05, and all micro-architectural parameters were significantly different between groups (p<0.05. Histological analyses showed almost all transplanted allogeneic bone was replaced by new bone, principally fibrous ossification, in the experimental group, which differed from the control group where little new bone formed.Our study demonstrated the feasibility of MSC-loaded allogenic mandibular scaffolds for the reconstruction of hemi-mandibular defects. Further studies are needed to test whether these results can be surpassed by the use of allogenic mandibular scaffolds loaded with a combination of MSCs and osteoinductive growth

  20. Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

    Institute of Scientific and Technical Information of China (English)

    Feng Yan; Wei Yue; Yue-lin Zhang; Guo-chao Mao; Ke Gao; Zhen-xing Zuo; Ya-jing Zhang; Hui Lu

    2015-01-01

    In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffoldin vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hi-tosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial ifbrillary acidic protein and a low level of expression of neuron-spe-ciifc enolase were visible in BrdU-labeled bone marrow mesenchymal stem cells. These ifndings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chi-tosan-collagen scaffold has a neuroprotective effect following ischemic stroke.

  1. Study of Carbon Nano-Tubes Effects on the Chondrogenesis of Human Adipose Derived Stem Cells in Alginate Scaffold

    Directory of Open Access Journals (Sweden)

    Ali Valiani

    2014-01-01

    Full Text Available Background: Osteoarthritis is one of the most common diseases in middle-aged populations in the World and could become the fourth principal cause of disability by the year 2020. One of the critical properties for cartilage tissue engineering (TE is the ability of scaffolds to closely mimic the extracellular matrix and bond to the host tissue. Therefore, TE has been presented as a technique to introduce the best combination of cells and biomaterial scaffold and to stimulate growth factors to produce a cartilage tissue resembling natural articular cartilage. The aim of study is to improve differentiation of adipose derived stem cells (ADSCs into chondrocytes in order to provide a safe and modern treatment for patients suffering from cartilage damages. Methods: After functionalization, dispersions and sterilizing carbon nano-tubes (CNTs, a new type of nanocomposite gel was prepared from water-soluble CNTs and alginate. ADSCs seeded in 1.5% alginate scaffold and cultured in chondrogenic media with and without transforming growth factor-β1 (TGF-β1 for 7 and 14 days. The genes expression of sex determining region Y-box 9 (SOX9, types II and X collagens was assessed by real-time polymerase chain reaction and the amount of aggrecan (AGC and type I collagen was measured by ELISA. Results: Our findings showed that the expression of essential cartilage markers, SOX9, type II collagen and AGC, in differentiated ADSCs at the concentration of 1 μg/ml CNTs in the presence of TGF-β1 were significantly increased in comparison with the control group (P < 0.001. Meanwhile, type X collagen expression and also type I collagen production were significantly decreased (P < 0.001. Conclusions: The results showed that utilized three-dimensional scaffold had a brilliant effect in promoting gene expression of chondrogenesis.

  2. The effect of Cu(II)-loaded brushite scaffolds on growth and activity of osteoblastic cells.

    Science.gov (United States)

    Ewald, Andrea; Käppel, Christine; Vorndran, Elke; Moseke, Claus; Gelinsky, Michael; Gbureck, Uwe

    2012-09-01

    Bone substitute materials such as calcium phosphate cements (CPC) are frequently used as growth factor carriers for the stimulation of osteoblast-formation around an implant. However, biological modification based on delicate protein factors like extracellular matrix proteins or growth factors is subject to a number of shortcomings like the need for storage below room temperature and cost of production. The aim of this study was to investigate ionic modification as an alternative bioinorganic route for implant modification. Although it is known that Cu(II) plays a role in angiogenesis and bone formation, not all involved processes are well understood yet. In this study the in vitro effect of Cu(II) on growth and activity of osteoblastic cells seeded on brushite (CaHPO(4) · 2 H(2) O) scaffolds as well as on glass discs was investigated. The results show that Cu(II) enhances cell activity and proliferation of osteoblastic cells on CPC and furthermore affects the expression of several bone specific proteins such as bone sialo protein or osteocalcin. Therefore, the modification of CPC with Cu(II) may offer a promising alternative to protein based modification to stimulate cellular activity for an improved bone healing.

  3. Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model.

    Science.gov (United States)

    Kang, Sun-Woong; Son, Sun-Mi; Lee, Jae-Sun; Lee, Eung-Seok; Lee, Kwon-Yong; Park, Sang-Guk; Park, Jung-Ho; Kim, Byung-Soo

    2006-09-01

    The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering

  4. Adhesion and proliferation of adipose derived mesenchymal stromal cells on chitosan scaffolds with different degree of deacetylation

    Directory of Open Access Journals (Sweden)

    Rogulska O. Yu.

    2014-03-01

    Full Text Available Aim. Selection of the optimal scaffold for the creation of tissue engineering constructs is a key challenge of biotechnology. In this study we investigated the biocompatibility of human adipose derived mesenchymal stromal cells (MSCs within the three-dimensional matrices based on the chitosan with a different degree of deacetylation. Methods. MSCs were seeded on the chitosan scaffolds by a perfusion method and cultured for 7 days. The morphology, viability, metabolic activity and distribution of the cells within the matrices were analyzed. Results. The level of MSCs adhesion to the surface of the chitosan scaffolds with low degree of deacetylation (67 % was insignificant, the cells were round and formed aggregates. In the chitosan scaffolds with a high degree of deacetylation (82 % the cells attached to the surface of matrices, were able to spread and proliferate. Conclusions. The chitosan scaffolds with a high degree of deacetylation and the human adipose derived MSCs can be used for the creation of bioengineered structures.

  5. Reduced liver cell death using an alginate scaffold bandage: a novel approach for liver reconstruction after extended partial hepatectomy.

    Science.gov (United States)

    Shteyer, Eyal; Ben Ya'acov, Ami; Zolotaryova, Lidia; Sinai, Avital; Lichtenstein, Yoav; Pappo, Orit; Kryukov, Olga; Elkayam, Tsiona; Cohen, Smadar; Ilan, Yaron

    2014-07-01

    Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank=0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (Palginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.

  6. Polyhydroxyalkanoates: waste glycerol upgrade into electrospun fibrous scaffolds for stem cells culture

    OpenAIRE

    Canadas, Raphael Faustino; Cavalheiro, João M. B. T.; Guerreiro, João D. T.; Almeida, M. Catarina M. D. de; Pollet, Eric; Silva, Cláudia Lobato da; Fonseca, M.M.R. da; Ferreira, Frederico Castelo

    2014-01-01

    This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic t...

  7. Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

    Directory of Open Access Journals (Sweden)

    Ken Ye

    Full Text Available Infrapatellar fat pad adipose stem cells (IPFP-ASCs have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

  8. The performance of dental pulp stem cells on nanofibrous PCL/gelatin/nHA scaffolds.

    NARCIS (Netherlands)

    Yang, X.; Yang, F.; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2010-01-01

    The aim of current study is to investigate the in vitro and in vivo behavior of dental pulp stem cells (DPSCs) seeded on electrospun poly(epsilon-caprolactone) (PCL)/gelatin scaffolds with or without the addition of nano-hydroxyapatite (nHA). For the in vitro evaluation, DNA content, alkaline phosph

  9. The performance of human dental pulp stem cells on different three-dimensional scaffold materials.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Kuppevelt, A.H.M.S.M. van; Daamen, W.F.; Bian, Z.; Jansen, J.A.

    2006-01-01

    The aim of this study was to investigate the in vitro and in vivo behavior of human dental pulp stem cells (DPSCs) isolated from impacted third molars, when seeded onto different 3-dimensional (3-D) scaffold materials: i.e. a spongeous collagen, a porous ceramic, and a fibrous titanium mesh. Scaffol

  10. In vitro evaluation of the compatibility of a novel collagen-heparan sulfate biological scaffold with olfactory ensheathing cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zhou-ping; YANG Jie; PAN Deng-ji; LIU Na; LI Zai-wang; XIE Xue-wei; CHEN Yun; SHI Yuan-hong; ZENG Wen-gao; WANG Shu-xin; CHEN Juan

    2010-01-01

    Background Stroke and traumatic injury to the nerve system may trigger axonal destruction and the formation of scar tissue, cystic cavitations and physical gaps.Olfactory ensheathing cells (OECs) can secrete neurotrophic factors to promote neurite growth and thus act as a prime candidate for autologous transplantation.Biological scaffolds can provide a robust delivery vehicle to injured nerve tissue for neural cell transplantation strategies, owing to the porous three-dimensional structures (3D).So transplantation of the purposeful cells seeded scaffolds may be a promising method for nerve tissue repair.This study aimed to evaluate the compatibility of a novel collagen-heparan sulfate biological scaffold with olfactory ensheathing cells in vitro.Methods Collagen-heparan sulfate (CHS) biological scaffolds were made, and then the scaffolds and OECs were co-cultured in vitro.The viability of OECs was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay at days 1, 3, 5 and 7.Statistical analysis was evaluated by student's t test.Significance was accepted at P <0.05.OECs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and the CFSE-labeled OECs were seeded into CHS scaffolds.The attachment and growth of OECs in CHS scaffolds were observed and traced directly by fluorescent microscopy and environmental scanning electron microscope (ESEM).Results CHS biological scaffolds had steady porous 3D structures and no cytotoxicity to OECs (F=0.14, P=0.9330).CHS biological scaffolds were good bridging materials for OECs attachment and proliferation, and they promoted the axonal growth.Conclusion The compatibility of CHS biological scaffolds with OECs is pretty good and CHS biological scaffold is a promising cell carrier for the implantation of OECs in nerve tissue bioengineering.

  11. Evaluation of biodegradable elastic scaffolds made of anionic polyurethane for cartilage tissue engineering.

    Science.gov (United States)

    Tsai, Meng-Chao; Hung, Kun-Che; Hung, Shih-Chieh; Hsu, Shan-hui

    2015-01-01

    Biodegradable polyurethane (PU) was synthesized by a water-based process. The process rendered homogenous PU nanoparticles (NPs). Spongy PU scaffolds in large dimensions were obtained by freeze-drying the PU NP dispersion. The spongy scaffolds were characterized in terms of the porous structure, wettability, mechanical properties, degradation behavior, and degradation products. The capacity as cartilage tissue engineering scaffolds was evaluated by growing chondrocytes and mesenchymal stem cells (MSCs) in the scaffolds. Scaffolds made from the PU dispersion had excellent hydrophilicity, porosity, and water absorption. Examination by micro-computed tomography confirmed that PU scaffolds had good pore interconnectivity. The degradation rate of the scaffolds in phosphate buffered saline was much faster than that in papain solution or in deionized water at 37°C. The biodegradable PU appeared to be degraded via the cleavage of ester linkage The intrinsic elastic property of PU and the gyroid-shape porous structure of the scaffolds may have accounted for the outstanding strain recovery (87%) and elongation behavior (257%) of the PU scaffolds, compared to conventional poly(d,l-lactide) (PLA) scaffolds. Chondrocytes were effectively seeded in PU scaffolds without pre-wetting. They grew better and secreted more glycosaminoglycan in PU scaffolds vs. PLA scaffolds. Human MSCs showed greater chondrogenic gene expression in PU scaffolds than in PLA scaffolds after induction. Based on the favorable hydrophilicity, elasticity, and regeneration capacities, the novel biodegradable PU scaffolds may be superior to the conventional biodegradable scaffolds in cartilage tissue engineering applications.

  12. Acellular Lung Scaffolds Direct Differentiation of Endoderm to Functional Airway Epithelial Cells: Requirement of Matrix-Bound HS Proteoglycans

    Directory of Open Access Journals (Sweden)

    Sharareh Shojaie

    2015-03-01

    Full Text Available Efficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

  13. Systems metabolic engineering of microorganisms to achieve large-scale production of flavonoid scaffolds.

    Science.gov (United States)

    Wu, Junjun; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-10-20

    Flavonoids possess pharmaceutical potential due to their health-promoting activities. The complex structures of these products make extraction from plants difficult, and chemical synthesis is limited because of the use of many toxic solvents. Microbial production offers an alternate way to produce these compounds on an industrial scale in a more economical and environment-friendly manner. However, at present microbial production has been achieved only on a laboratory scale and improvements and scale-up of these processes remain challenging. Naringenin and pinocembrin, which are flavonoid scaffolds and precursors for most of the flavonoids, are the model molecules that are key to solving the current issues restricting industrial production of these chemicals. The emergence of systems metabolic engineering, which combines systems biology with synthetic biology and evolutionary engineering at the systems level, offers new perspectives on strain and process optimization. In this review, current challenges in large-scale fermentation processes involving flavonoid scaffolds and the strategies and tools of systems metabolic engineering used to overcome these challenges are summarized. This will offer insights into overcoming the limitations and challenges of large-scale microbial production of these important pharmaceutical compounds.

  14. Concise review: tailoring bioengineered scaffolds for stem cell applications in tissue engineering and regenerative medicine.

    Science.gov (United States)

    Cosson, Steffen; Otte, Ellen A; Hezaveh, Hadi; Cooper-White, Justin J

    2015-02-01

    The potential for the clinical application of stem cells in tissue regeneration is clearly significant. However, this potential has remained largely unrealized owing to the persistent challenges in reproducibly, with tight quality criteria, and expanding and controlling the fate of stem cells in vitro and in vivo. Tissue engineering approaches that rely on reformatting traditional Food and Drug Administration-approved biomedical polymers from fixation devices to porous scaffolds have been shown to lack the complexity required for in vitro stem cell culture models or translation to in vivo applications with high efficacy. This realization has spurred the development of advanced mimetic biomaterials and scaffolds to increasingly enhance our ability to control the cellular microenvironment and, consequently, stem cell fate. New insights into the biology of stem cells are expected to eventuate from these advances in material science, in particular, from synthetic hydrogels that display physicochemical properties reminiscent of the natural cell microenvironment and that can be engineered to display or encode essential biological cues. Merging these advanced biomaterials with high-throughput methods to systematically, and in an unbiased manner, probe the role of scaffold biophysical and biochemical elements on stem cell fate will permit the identification of novel key stem cell behavioral effectors, allow improved in vitro replication of requisite in vivo niche functions, and, ultimately, have a profound impact on our understanding of stem cell biology and unlock their clinical potential in tissue engineering and regenerative medicine.

  15. Modification of the diphenylamine assay for cell quantification in three-dimensional biodegradable polymeric scaffolds.

    Science.gov (United States)

    Pham, Edward A; Ho, Won Jin; Kamei, Daniel T; Wu, Benjamin M

    2010-02-01

    As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects. Thus, additional steps are required to ensure complete isolation of cells from the 3D scaffold. The diphenylamine assay isolates cellular DNA, degrades the polymeric matrix materials, and reacts with the DNA to yield a colorimetric response. Thus, we report here a practical modification of the diphenylamine assay and show that the assay quantifies cells in 3D polyester scaffolds reliably and reproducibly as long as the necessary amount of the acidic working reagent is present. Our study also demonstrates that the sensitivity of the assay can be optimized by controlling the dimensions of the sampling volume. Overall, the DPA assay offers an attractive solution for challenges associated with 3D cell quantification.

  16. Development of three-dimensional collagen scaffolds with controlled architecture for cell migration studies using breast cancer cell lines.

    Science.gov (United States)

    Campbell, Jonathan J; Husmann, Anke; Hume, Robert D; Watson, Christine J; Cameron, Ruth E

    2017-01-01

    Cancer is characterized by cell heterogeneity and the development of 3D in vitro assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues in vitro and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.

  17. Collagen-GAG Scaffolds Grafted Onto Myocardial Infarcts in a Rat Model: A Delivery Vehicle for Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Z. XIANG; R. LIAO; M. KELLY; M. SPECTOR

    2005-01-01

    @@ 1 Introduction The objective of the present study was to investigate the response of rat myocardial scar tissue to type Ⅰ collagen-glycosaminoglycan (GAG) tissue engineering scaffolds, and to assess the feasibility of using a collagenGAG scaffold as a delivery vehicle for bone marrow-derived mesenchymal stem cells. The benefits of employing the collagen-GAG scaffold for this application include the following: (1) the large surface area of the three-dimensional sponge-like material allows for the delivery of a high cell density to the infarct site; (2) the scaffold allows for the localization and retention of the cells at the site of implantation; (3) the tissue response to the scaffold may promote angiogenesis at the implant site.

  18. The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration.

    Science.gov (United States)

    Tabesh, H; Amoabediny, Gh; Nik, N Salehi; Heydari, M; Yosefifard, M; Siadat, S O Ranaei; Mottaghy, K

    2009-02-01

    Spinal cord injury is very complicated, as there are factors in the body that inhibit its repair. Although regeneration of the mammalian central nervous system (CNS) was once thought to be impossible, studies over the past two decades have shown that axonal growth after spinal cord injury can occur when provided with the correct substratum. Traditionally, tissue transplantation or peripheral nerve grafting are used to repair damaged or diseased regions of the CNS, but donor shortage and immunological problems associated with infectious disease are often encountered. Fortunately, recent advances in neuroscience, cell culture, and biomaterials provide optimistic future using new treatments for nerve injuries. Biomaterial scaffold creates substrate within which cells are instructed to form a tissue or an organ in a highly controlled way. The principal function of a scaffold is to direct cell behavior such as migration, proliferation, differentiation, maintenance of phenotype, and apoptosis by facilitating sensing and responding to the environment via cell-matrix and cell-cell communications. Therefore, having such abilities provides scaffolds seeded with a special type of cell as an important part of tissue engineering and regenerative medicine which spinal cord regeneration is an example of. Nevertheless, the vast number of biodegradable synthetic and natural biopolymers makes choosing the right one very difficult. In this review article, it was tried to provide an inclusive survey of biopolymers seeded with Schwann cells (SCs) to be used for axonal regeneration in the nervous system.

  19. Cell-free scaffolds with different stiffness but same microstructure promote bone regeneration in rabbit large bone defect model.

    Science.gov (United States)

    Chen, Guobao; Yang, Li; Lv, Yonggang

    2016-04-01

    To promote bone healing, bone repair biomaterials are increasingly designed to incorporate growth factors. However, the impact of matrix mechanics of cell-free scaffold independent of microstructure on the osteogenic differentiation of endogenous osteoprogenitor cells orchestrating bone repair and regeneration remains not to be fully understood. In our recent study, three-dimensional (3D) scaffolds with different stiffness but same microstructure have been successfully fabricated by coating decellularized bone with collagen/hydroxyapatite (HA) mixture with different collagen rations. It has been demonstrated that the scaffold with optimal stiffness can induce the osteogenic differentiation of MSCs in vitro and in the subcutaneous tissue. The present in vivo study further investigated the repair efficiency of these scaffolds in a rabbit radius with a critical-sized segmental defect model and its potential mechanism. Micro-computed tomography (μ-CT), X-ray and histological analysis were carried out to evaluate the repair capacity of these scaffolds. The results demonstrated that the cell-free scaffold with optimal stiffness incorporation of endogenous osteoprogenitor cells significantly promoted the repair and reconstruction quality of mass bone defect. One of the crucial mechanisms was that hypoxia and stromal cell-derived factor-1α (SDF-1α) mediated mesenchymal stem cells (MSCs) migration by which matrix mechanics exerted influence on bone fracture healing. These findings suggested that only modulating the matrix stiffness of cell-free scaffold can be one of the most attractive strategies for promoting the progression of bone healing.

  20. Pore architecture and cell viability on freeze dried 3D recombinant human collagen-peptide (RHC)–chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Zhou, Aimei; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Zhong, Zhaocai [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-04-01

    Pore architecture of 3D scaffolds used in tissue engineering plays a critical role in the maintenance of cell survival, proliferation and further promotion of tissue regeneration. We investigated the pore size and structure, porosity, swelling as well as cell viability of a series of recombinant human collagen-peptide–chitosan (RHCC) scaffolds fabricated by lyophilization. In this paper, freezing regime containing a final temperature of freezing (T{sub f}) and cooling rates was applied to obtain scaffolds with pore size ranging from 100 μm to 120 μm. Other protocols of RHC/chitosan suspension concentration and ratio modification were studied to produce more homogenous and appropriate structural scaffolds. The mean pore size decreased along with the decline of T{sub f} at a slow cooling rate of 0.7 °C/min; a more rapid cooling rate under 5 °C/min resulted to a smaller pore size and more homogenous microstructure. High concentration could reduce pore size and lead to thick well of scaffold, while improved the ratio of RHC, lamellar and fiber structure coexisted with cellular pores. Human umbilical vein endothelial cells (HUVECs) were seeded on these manufactured scaffolds, the cell viability represented a negative correlation to the pore size. This study provides an alternative method to fabricate 3D RHC–chitosan scaffolds with appropriate pores for potential tissue engineering. - Highlights: • Fabrication of recombinant human collagen-chitosan scaffolds by freezing drying • Influence of freeze drying protocols on lyophilized scaffolds • Pore size, microstructure, porosity, swelling and cell viability were compared. • The optimized porous scaffold is suitable for cell (HUVEC) seeding.

  1. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Science.gov (United States)

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-05-24

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm).

  2. In vitro evaluation of three different biomaterials as scaffolds for canine mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Oduvaldo Câmara Marques Pereira-Junior

    2013-05-01

    Full Text Available PURPOSE: To evaluate in vitro ability the of three different biomaterials - purified hydroxyapatite, demineralized bone matrix and castor oil-based polyurethane - as biocompatible 3D scaffolds for canine bone marrow mesenchymal stem cell (MSC intending bone tissue engineering. METHODS: MSCs were isolated from canine bone marrow, characterized and cultivated for seven days with the biomaterials. Cell proliferation and adhesion to the biomaterial surface were evaluated by scanning electron microscopy while differentiation into osteogenic lineage was evaluated by Alizarin Red staining and Sp7/Osterix surface antibody marker. RESULTS: The biomaterials allowed cellular growth, attachment and proliferation. Osteogenic differentiation occurred in the presence of hydroxyapatite, and matrix deposition commenced in the presence of the castor oil-based polyurethane. CONCLUSION: All the tested biomaterials may be used as mesenchymal stem cell scaffolds in cell-based orthopedic reconstructive therapy.

  3. Fabrication and Cell Responsive Behavior of Macroporous PLLA/Gelatin Composite Scaffold with Hierarchical Micro-Nano Pore Structure

    Directory of Open Access Journals (Sweden)

    Kedong Song

    2015-03-01

    Full Text Available Scaffolds providing a 3D environment which can effectively promote the adhesion, proliferation and differentiation of cells are crucial to tissue regeneration. In this study, the poly-l-lactic acid (PLLA scaffold with hierarchical pore structural was fabricated via two-step thermally induced phase separation (TIPS. To mimic both physical architecture and chemical composite of natural bone extracellular matrix (ECM, gelatin fibers were introduced into the pores of PLLA scaffolds and formed 3D network structure via TIPS. Human adipose tissue-derived stem cells (ADSCs were harvested and seeded into PLLA/gel hybrid scaffolds and cultured in vitro for biocompatibility assay. The surface morphology, porosity and compressive modulus of scaffolds were characterized by scanning electron microscopy (SEM, density analysis and compression test respectively. The results showed that hybrid scaffolds had high porosity (91.62%, a good compressive modulus (2.79 ± 0.20 MPa, nanometer fibers (diameter around 186.39~354.30 nm and different grades of pore size from 7.41 ± 2.64 nm to 387.94 ± 102.48 nm. The scaffolds with mild hydrolysis by NaOH were modified by 1-ethyl-3-(3-dimethyl ami-nopropyl carbodiimide/N-hydroxysuccinimide (EDC/NHS. Gelatin was performed onto PLLA scaffold via TIPS aiming at enhancement cell-material interaction. In comparison with PLLA scaffold, the PLLA/gel scaffold had better biological performance and the mechanical properties because the gelatin fibers homogeneously distributed in each pore of PLLA scaffold and formed 3D network structure.

  4. Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors.

    Science.gov (United States)

    Sladkova, Martina; Palmer, Michael; Öhman, Caroline; Alhaddad, Rawan Jaragh; Esmael, Asmaa; Engqvist, Håkan; de Peppo, Giuseppe Maria

    2016-12-01

    Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications.

  5. The evaluation of physical properties and in vitro cell behavior of PHB/PCL/sol-gel derived silica hybrid scaffolds and PHB/PCL/fumed silica composite scaffolds.

    Science.gov (United States)

    Ding, Yaping; Yao, Qingqing; Li, Wei; Schubert, Dirk W; Boccaccini, Aldo R; Roether, Judith A

    2015-12-01

    PHB/PCL/sol-gel derived silica hybrid scaffolds (P5S1S) and PHB/PCL/fumed silica composite scaffolds (P5S1N) with a 5:1 organic/inorganic ratio were fabricated through a combination of electrospinning and sol-gel methods and dispersion electrospinning, respectively. In contrast to the silica nanoparticle aggregates appearing on the fiber surface of P5S1N, smooth and uniform fibers were obtained for P5S1S. The fiber diameter distribution, tensile strength, thermal gravimetric analysis (TGA), and cellular behavior of both types of scaffolds were characterized and studied. The tensile strength results and TGA indicated that the interfacial interaction between the organic and the inorganic phase was enhanced in P5S1S over the nanocomposite scaffolds, and cells exhibited significantly higher alkaline phosphate activity (ALP) for P5S1S, which makes P5S1S hybrid scaffolds candidate materials for bone tissue engineering applications.

  6. The Effects of Scaffold Remnants in Decellularized Tissue Engineered Cardiovascular Constructs on the Recruitment of Blood Cells.

    Science.gov (United States)

    Sanders, Bart; Driessen-Mol, Anita; Bouten, Carlijn; Baaijens, Frank

    2017-03-17

    Decellularized tissue engineered heart valves (DTEHVs) showed remarkable results in translational animal models, leading to recellularization within hours after implantation. This is crucial to enable tissue remodeling. To investigate if the presence of scaffold remnants prior to implantation is responsible for the fast recellularization of DTEHVs, an in vitro mesofluidic system was used. Human granulocyte and agranulocyte fractions were isolated, stained, brought back in suspension, and implemented in the system. Three different types of biomaterials were exposed to the circulating blood cells, consisting of decellularized tissue engineered constructs (DTEC) with or without scaffold remnants, or only bare scaffold. After 5 hours of testing, the granulocyte fraction was depleted faster from the circulation than the agranulocyte fraction. However, only the granulocytes infiltrated into the DTEC with scaffold, migrating towards the scaffold remnants. The agranulocyte population, on the other hand, was only observed on the outer surface. Active cell infiltration was associated with increased levels of MMP-1 secretion in the DTEC including scaffold remnants. Pro-inflammatory cytokines such as IL-1α, IL-6 and TNF-α were significantly upregulated in the DTEC without scaffold remnants. These results indicate that scaffold remnants can influence the immune response in DTEC, being responsible for rapid cell infiltration.

  7. Asynchronous Inflammation and Myogenic Cell Migration Limit Muscle Tissue Regeneration Mediated by a Cellular Scaffolds

    Science.gov (United States)

    2015-02-11

    over two-times that observed with muscle grafts, but they appeared to be less active, as gene expression of pro- and anti- inflammatory cytokines ( TNF -α...injury) the inflammatory and myogenic response to the muscle scaffold [16], which relies solely on host cell migration for regeneration [18]. Vital...cells [37] to induce myogenesis. Following injury, the type of the inflammatory response and the significance of transition from pro- to an anti

  8. TATVHL peptide-grafted alginate/poly(γ-glutamic acid) scaffolds with inverted colloidal crystal topology for neuronal differentiation of iPS cells.

    Science.gov (United States)

    Kuo, Yung-Chih; Chung, Chiu-Yen

    2012-12-01

    The neuronal differentiation of induced pluripotent stem (iPS) cells in scaffolding biomaterials is an emerging issue in nervous regeneration and repair. This study presents the production of neuron-lineage cells from iPS cells in inverted colloidal crystal (ICC) scaffolds comprising alginate, poly(γ-glutamic acid) (γ-PGA), and TATVHL peptide. The ability of iPS cells to differentiate toward neurons in the constructs was demonstrated by flow-cytometeric sorting and immunochemical staining. The results revealed that hexagonally arrayed microspheres molded alginate/γ-PGA hydrogel into ICC topology with adequate interconnected pores. An increase in the quantity of surface TATVHL peptide enhanced the atomic ratio of nitrogen and the adhesion efficiency of iPS cells in constructs. However, the effect of TATVHL peptide on the viability of iPS cells was insignificant. The adhesion and viability of iPS cells in ICC constructs was higher than those in freeform ones. TATVHL peptide raised the percentage of β III tubulin-identified cells differentiating from iPS cells, indicating that TATVHL peptide stimulated the neuronal development in alginate/γ-PGA ICC constructs. TATVHL peptide-grafted alginate/γ-PGA ICC scaffolds can be promising for establishing nerve tissue from iPS cells.

  9. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  10. The Three-Dimensional Collagen Scaffold Improves the Stemness of Rat Bone Marrow Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Sufang Han; Yannan Zhao; Zhifeng Xiao; Jin Han; Bing Chen; Lei Chen; Jianwu Dai

    2012-01-01

    Mesenchymal stem cells (MSCs) show the great promise for the treatment of a variety of diseases because of their self-renewal and multipotential abilities.MSCs are generally cultured on two-dimensional (2D) substrate in vitro.There are indications that they may simultaneously lose their stemness and multipotentiality as the result of prolonged 2D culture.In this study,we used three-dimensional (3D) collagen scaffolds as rat MSCs carrier and compared the properties of MSCs on 3D collagen scaffolds with monolayer cultured MSCs.The results demonstrated that collagen scaffolds were suitable for rat MSCs adherence and proliferation.More importantly,compared to MSCs under 2D culture,3D MSCs significantly maintained higher expression levels of stemness genes (Oct4,Sox2,Rex-1 and Nanog),yielded high frequencies of colony-forming units-fibroblastic (CFU-F) and showed enhanced osteogenic and adipogenic differentiation efficiency upon induction.Thus,3D collagen scaffolds may be beneficial for expanding rat MSCs while maintaining the stem cell properties in vitro.

  11. In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds.

    Science.gov (United States)

    Sangsanoh, Pakakrong; Waleetorncheepsawat, Suchada; Suwantong, Orawan; Wutticharoenmongkol, Patcharaporn; Weeranantanapan, Oratai; Chuenjitbuntaworn, Boontharika; Cheepsunthorn, Poonlarp; Pavasant, Prasit; Supaphol, Pitt

    2007-05-01

    The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.

  12. Strategies for bioengineered scaffolds that support adipose stem cells in regenerative therapies.

    Science.gov (United States)

    Clevenger, Tracy N; Luna, Gabriel; Fisher, Steven K; Clegg, Dennis O

    2016-09-01

    Regenerative medicine possesses the potential to ameliorate damage to tissue that results from a vast range of conditions, including traumatic injury, tumor resection and inherited tissue defects. Adult stem cells, while more limited in their potential than pluripotent stem cells, are still capable of differentiating into numerous lineages and provide feasible allogeneic and autologous treatment options for many conditions. Adipose stem cells are one of the most abundant types of stem cell in the adult human. Here, we review recent advances in the development of synthetic scaffolding systems used in concert with adipose stem cells and assess their potential use for clinical applications.

  13. Flow perfusion enhances the calcified matrix deposition of marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds.

    Science.gov (United States)

    Sikavitsas, Vassilios I; Bancroft, Gregory N; Lemoine, Jeremy J; Liebschner, Michael A K; Dauner, Martin; Mikos, Antonios G

    2005-01-01

    In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 microm fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 x 10(5) cells in 300 microl, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.

  14. The effect of porosity on cell ingrowth into accurately defined, laser-made, polylactide-based 3D scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Danilevicius, Paulius; Georgiadi, Leoni [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Pateman, Christopher J.; Claeyssens, Frederik [Kroto Research Institute, Department of Materials Science and Engineering, University of Sheffield, Broad Lane, Sheffield S3 7HQ (United Kingdom); Chatzinikolaidou, Maria, E-mail: mchatzin@materials.uoc.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece); Department of Materials Science and Technology, University of Crete, PO Box 2208, 71303 Heraklion (Greece); Farsari, Maria, E-mail: mfarsari@iesl.forth.gr [Foundation for Research and Technology Hellas (FORTH), Institute of Electronic Structure and Laser (IESL), N Plastira 100, 70013 Heraklion (Greece)

    2015-05-01

    Highlights: • We studied the porosity of laser-made 3D scaffolds on MC3T3-E1 pre-osteoblastic cells. • We made polylactide 3D scaffolds with pores 25–110 μm. - Abstract: The aim of this study is to demonstrate the accuracy required for the investigation of the role of solid scaffolds’ porosity in cell proliferation. We therefore present a qualitative investigation into the effect of porosity on MC3T3-E1 pre-osteoblastic cell ingrowth of three-dimensional (3D) scaffolds fabricated by direct femtosecond laser writing. The material we used is a purpose made photosensitive pre-polymer based on polylactide. We designed and fabricated complex, geometry-controlled 3D scaffolds with pore sizes ranging from 25 to 110 μm, representing porosities 70%, 82%, 86%, and 90%. The 70% porosity scaffolds did not support cell growth initially and in the long term. For the other porosities, we found a strong adhesion of the pre-osteoblastic cells from the first hours after seeding and a remarkable proliferation increase after 3 weeks and up to 8 weeks. The 86% porosity scaffolds exhibited a higher efficiency compared to 82% and 90%. In addition, bulk material degradation studies showed that the employed, highly-acrylated polylactide is degradable. These findings support the potential use of the proposed material and the scaffold fabrication technique in bone tissue engineering.

  15. Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells

    Science.gov (United States)

    Weber, L.; Langer, M.; Tavella, S.; Ruggiu, A.; Peyrin, F.

    2016-05-01

    In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite©), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions.

  16. Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

    DEFF Research Database (Denmark)

    Bjerre, Lea; Bünger, Cody; Baatrup, Anette;

    2011-01-01

    of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static......Bone grafts are widely used in orthopaedic reconstructive surgery, but harvesting of autologous grafts is limited due to donor site complications. Bone tissue engineering is a possible alternative source for substitutes, and to date, mainly small scaffold sizes have been evaluated. The aim...... culture for up to 21 days and analysed for cell distribution and osteogenic differentiation using histological stainings, alkaline phosphatase activity assay, and real-time RT-PCR on bone markers. We found that the number of cells was higher during static culture at most time points and that the final...

  17. Biodegradable Cell-Seeded Nanofiber Scaffolds for Neural Repair

    Directory of Open Access Journals (Sweden)

    Karen C. Cheung

    2011-10-01

    Full Text Available Central and peripheral neural injuries are traumatic and can lead to loss of motor and sensory function, chronic pain, and permanent disability. Strategies that bridge the site of injury and allow axonal regeneration promise to have a large impact on restoring quality of life for these patients. Engineered materials can be used to guide axonal growth. Specifically, nanofiber structures can mimic the natural extracellular matrix, and aligned nanofibers have been shown to direct neurite outgrowth and support axon regeneration. In addition, cell-seeded scaffolds can assist in the remyelination of the regenerating axons. The electrospinning process allows control over fiber diameter, alignment, porosity, and morphology. Biodegradable polymers have been electrospun and their use in tissue engineering has been demonstrated. This paper discusses aspects of electrospun biodegradable nanofibers for neural regeneration, how fiber alignment affects cell alignment, and how cell-seeded scaffolds can increase the effectiveness of such implants.

  18. Interfacing Sca-1pos Mesenchymal Stem Cells with Biocompatible Scaffolds with Different Chemical Composition and Geometry

    Directory of Open Access Journals (Sweden)

    G. Forte

    2009-01-01

    Full Text Available An immortalized murine mesenchymal stem cell line (mTERT-MSC enriched for Linneg/Sca-1pos fraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 μm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.

  19. Enhanced osteogenic differentiation of mesenchymal stem cells on poly(L-lactide) nanofibrous scaffolds containing carbon nanomaterials.

    Science.gov (United States)

    Duan, Shun; Yang, Xiaoping; Mei, Fang; Tang, Yan; Li, Xiaoli; Shi, Yuzhou; Mao, Jifu; Zhang, Hongquan; Cai, Qing

    2015-04-01

    Carbon nanomaterials (CNMs), such as carbon nanotube (CNT) and graphene, are highlighted in bone regeneration because of their osteoinductive properties. Their combinations with nanofibrous polymeric scaffolds, which mimic the morphology of natural extracellular matrix of bone, arouse keen interest in bone tissue engineering. To this end, CNM were incorporated into nanofibrous poly(L-lactic acid) scaffolds by thermal-induced phase separation. The CNM-containing composite nanofibrous scaffolds were biologically evaluated by both in vitro co-culture of bone mesenchymal stem cells (BMSCs) and in vivo implantation. The nanofibrous structure itself demonstrated significant enhancement in cell adhesion, proliferation and oseogenic differentiation of BMSCs, and with the incorporation of CNM, the composite nanofibrous scaffolds further promoted osteogenic differentiation of BMSCs significantly. Between the two CNMs, graphene showed stronger effect in promoting osteogenic differentiation of BMSCs than CNT. The results of in vivo experiments revealed that the composite nanofibrous scaffolds had both good biocompatibility and strong ability in inducing osteogenesis. CNMs could remarkably enhance the expression of osteogenesis-related proteins as well as the formation of type I collagen. Similarly, the graphene-containing composite nanofibrous scaffolds demonstrated the strongest effect on inducing osteogenesis in vivo. These findings demonstrated that CNM-containing composite nanofibrous scaffolds were obviously more efficient in promoting osteogenesis than pure polymeric scaffolds.

  20. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P R Anil [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Varma, H K [Bioceramics Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Kumary, T V [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India)

    2007-03-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function.

  1. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering.

    Science.gov (United States)

    Anil Kumar, P R; Varma, H K; Kumary, T V

    2007-03-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function.

  2. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    Science.gov (United States)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS – which was released from scaffolds quickly – significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  3. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro.

    Science.gov (United States)

    Tamaddon, M; Burrows, M; Ferreira, S A; Dazzi, F; Apperley, J F; Bradshaw, A; Brand, D D; Czernuszka, J; Gentleman, E

    2017-03-03

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  4. Application of visible light-based projection stereolithography for live cell-scaffold fabrication with designed architecture.

    Science.gov (United States)

    Lin, Hang; Zhang, Dongning; Alexander, Peter G; Yang, Guang; Tan, Jian; Cheng, Anthony Wai-Ming; Tuan, Rocky S

    2013-01-01

    One-step scaffold fabrication with live cell incorporation is a highly desirable technology for tissue engineering and regeneration. Projection stereolithography (PSL) represents a promising method owing to its fine resolution, high fabrication speed and computer-aided design (CAD) capabilities. However, the majority of current protocols utilize water-insoluble photoinitiators that are incompatible with live cell-fabrication, and ultraviolet (UV) light that is damaging to the cellular DNA. We report here the development of a visible light-based PSL system (VL-PSL), using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the initiator and polyethylene glycol diacrylate (PEGDA) as the monomer, to produce hydrogel scaffolds with specific shapes and internal architectures. Furthermore, live human adipose-derived stem cells (hADSCs) were suspended in PEGDA/LAP solution during the PSL process, and were successfully incorporated within the fabricated hydrogel scaffolds. hADSCs in PEG scaffolds showed high viability (>90%) for up to 7 days after fabrication as revealed by Live/Dead staining. Scaffolds with porous internal architecture retained higher cell viability and activity than solid scaffolds, likely due to increased oxygen and nutrients exchange into the interior of the scaffolds. The VL-PSL should be applicable as an efficient and effective tissue engineering technology for point-of-care tissue repair in the clinic.

  5. Bacterial scaffolds assemble novel higher-order complexes to reengineer eukaryotic cell processes.

    Science.gov (United States)

    Lesser, Cammie F; Leong, John M

    2011-07-05

    Many microbial pathogens use specialized secretion systems to inject proteins referred to as effectors directly into eukaryotic host cells. These effectors directly target various eukaryotic signaling pathways and cellular processes, often by mimicking the activity of host cell proteins. Effectors of pathogenic Escherichia coli and Salmonella typhimurium can also act as molecular scaffolds that not only recruit but also directly regulate the activity and localization of multiple eukaryotic proteins. By assembling and localizing disparate signaling pathways, the bacteria can reengineer host cell processes to generate novel processes not previously observed in eukaryotic cells.

  6. Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

    Science.gov (United States)

    Rogozhnikov, Dmitry; O’Brien, Paul J.; Elahipanah, Sina; Yousaf, Muhammad N.

    2016-12-01

    There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

  7. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds

    Science.gov (United States)

    Goldstein, A. S.; Juarez, T. M.; Helmke, C. D.; Gustin, M. C.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

  8. Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering.

    Science.gov (United States)

    Sumita, Yoshinori; Honda, Masaki J; Ohara, Takayuki; Tsuchiya, Shuhei; Sagara, Hiroshi; Kagami, Hideaki; Ueda, Minoru

    2006-06-01

    Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber mesh, although the success rate of tooth production is low. The present study was designed to compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks. After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively that a collagen sponge scaffold allows tooth production with a higher degree of success than polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.

  9. Hierarchical polymeric scaffolds support the growth of MC3T3-E1 cells.

    Science.gov (United States)

    Akbarzadeh, Rosa; Minton, Joshua A; Janney, Cara S; Smith, Tyler A; James, Paul F; Yousefi, Azizeh-Mitra

    2015-02-01

    Tissue engineering makes use of the principles of biology and engineering to sustain 3D cell growth and promote tissue repair and/or regeneration. In this study, macro/microporous scaffold architectures have been developed using a hybrid solid freeform fabrication/thermally induced phase separation (TIPS) technique. Poly(lactic-co-glycolic acid) (PLGA) dissolved in 1,4-dioxane was used to generate a microporous matrix by the TIPS method. The 3D-bioplotting technique was used to fabricate 3D macroporous constructs made of polyethylene glycol (PEG). Embedding the PEG constructs inside the PLGA solution prior to the TIPS process and subsequent extraction of PEG following solvent removal (1,4-dioaxane) resulted in a macro/microporous structure. These hierarchical scaffolds with a bimodal pore size distribution (300 μm) contained orthogonally interconnected macro-channels generated by the extracted PEG. The diameter of the macro-channels was varied by tuning the dispensing parameters of the 3D bioplotter. The in vitro cell culture using murine MC3T3-E1 cell line for 21 days demonstrated that these scaffolds could provide a favorable environment to support cell adhesion and growth.

  10. Enhanced photovoltaic performance of perovskite solar cells with mesoporous SiO2 scaffolds

    Science.gov (United States)

    Yu, Xiao; Chen, Si; Yan, Kai; Cai, Xin; Hu, Hsienwei; Peng, Ming; Chen, Buxin; Dong, Bin; Gao, Xue; Zou, Dechun

    2016-09-01

    We applied SiO2 nanoparticles as an alternative to mesoporous TiO2 or Al2O3 scaffolds and achieved power conversion efficiency (PCE) of up to 16.2%. Careful characterization of the effects of different scaffolds on device performance reveals that SiO2-based perovskite solar cells show much higher PCE due to the higher Voc and fill factor (FF), and the hysteresis for SiO2-based perovskite solar cell is less severe than for Al2O3-based perovskite solar cell. Time-correlated-single-photoncounting (TCSPC) luminescence decay and external quantum efficiency (EQE) tests provide further insights into the charge transfer behavior and light-harvesting characteristics of the proposed devices. Potential mechanisms of the observed phenomena are also suggested. Taken together, our results demonstrate that SiO2 nanoparticles may potentially replace mesoporous TiO2 or Al2O3 scaffolds in perovskite solar cells to achieve remarkably high PCE.

  11. Using Hydroxyapatite-Gelatin Scaffold Seeded with Bone Marrow Stromal Cells as a Bone Graft in Animal Model

    Directory of Open Access Journals (Sweden)

    Mahsoumeh Behruzi

    2016-11-01

    Full Text Available Background: Nowadays, composite scaffolds with some desired characteristics have a numerous applications in hard tissue engineering. In present study, the role of composite hydroxyapatite - gelatin was examined in both alone and coated by Bone Marrow Stromal Stem Cells (BMSCs conditions in the process of healing bone defects, reduction of time repair and the immune response of body by laboratory studies (in vitro and in vivo on the skull of adult rats as well. Materials and Methods: In present study, nano-hydroxyapatite powder and gelatin were used to provide nano-hydroxyapatite-gelatin scaffold, BMSCs were isolated by Flushing method. Fifteen adult male Wistar rats weighing 250-200 g were used. Studing groups included bone defect with hydroxyapatite-gelatin scaffold, bone defect with hydroxyapatite-gelatin with BMSCs and bone defects without scaffolding as a controlwhich were examined after a week and a month after surgery. MTT assay was used in order to evaluation of biocompatibility of scaffolds. To confirm the healing progress trend and the presence of inflammatory cells we used hematoxylin-eosin and we used Masson's trichrome staining in order to study of synthesis of collagen fibers. Results: The results of MTT showed that the scaffold has no toxic effects on stromal cells. The first signs of ossification in hydroxyapatite-gelatin with BMSCs cells group, appeared in the first week. However, in the fourth week, ossification was completed and the scaffold remaining was found as embedded islands in the spongy bone tissue. The greatest number of lymphocytes was observed in the experimental group after one week of planting scaffold. Conclusion: it seems that Hydroxyapatite-gelatin scaffold coated with BMSCs cells has a potential role in the healing process of bone and it can be suitable as a therapeutic strategy to repair extensive bone lesions.

  12. Influence of Fe3O4 Nanoparticles in Hydroxyapatite Scaffolds on Proliferation of Primary Human Fibroblast Cells

    Science.gov (United States)

    Maleki-Ghaleh, H.; Aghaie, E.; Nadernezhad, A.; Zargarzadeh, M.; Khakzad, A.; Shakeri, M. S.; Beygi Khosrowshahi, Y.; Siadati, M. H.

    2016-06-01

    Modern techniques for expanding stem cells play a substantial role in tissue engineering: the raw material that facilitates regeneration of damaged tissues and treats diseases. The environmental conditions and bioprocessing methods are the primary determinants of the rate of cultured stem cell proliferation. Bioceramic scaffolds made of calcium phosphate are effective substrates for optimal cell proliferation. The present study investigates the effects of two bioceramic scaffolds on proliferating cells in culture media. One scaffold was made of hydroxyapatite and the other was a mixture of hydroxyapatite and ferromagnetic material (Fe3O4 nanoparticles). Disk-shaped (10 mm × 2 mm) samples of the two scaffolds were prepared. Primary human fibroblast proliferation was 1.8- and 2.5-fold faster, respectively, when cultured in the presence of hydroxyapatite or ferrous nanoparticle/hydroxyapatite mixtures. Optical microscopy images revealed that the increased proliferation was due to enhanced cell-cell contact. The presence of magnetic Fe3O4 nanoparticles in the ceramic scaffolds significantly increased cell proliferation compared to hydroxyapatite scaffolds and tissue culture polystyrene.

  13. siRNA nanoparticle functionalization of nanostructured scaffolds enables controlled multilineage differentiation of stem cells

    DEFF Research Database (Denmark)

    Andersen, Morten Ø; Nygaard, Jens V; Burns, Jorge S;

    2010-01-01

    small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic......The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different...... differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas...

  14. Differentiation of Dental Pulp Stem Cells on Gutta-Percha Scaffolds

    OpenAIRE

    Liudi Zhang; Yingjie Yu; Christopher Joubert; George Bruder; Ying Liu; Chung-Chueh Chang; Marcia Simon; Walker, Stephen G.; Miriam Rafailovich

    2016-01-01

    Advances in treatment of tooth injury have shown that tooth regeneration from the pulp was a viable alternative of root canal therapy. In this study, we demonstrated that Gutta-percha, nanocomposites primarily used for obturation of the canal, are not cytotoxic and can induce differentiation of dental pulp stem cells (DPSC) in the absence of soluble mediators. Flat scaffolds were obtained by spin coating Si wafers with three Gutta-percha compounds: GuttaCore™, ProTaper™, and Lexicon™. The ima...

  15. A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

    Science.gov (United States)

    2014-10-01

    Scientific , Holliston, MA) syringe pump set at dispensing rate of 0.05 ml/hr. The syringe pump was placed on linear stage programmed to move to a...and new proposals on cell seeding nanofiber scaffolds are being developed. There is no scientific overlap with the current award. C...unaligned (C and E) and aligned (D and F) Matrigel ® coated nanofibers for 19 days and visualised using epifluorescence imaging. Neurons grown on aligned

  16. Parameterizing the Transport Pathways for Cell Invasion in Complex Scaffold Architectures

    Science.gov (United States)

    Ashworth, Jennifer C.; Mehr, Marco; Buxton, Paul G.; Best, Serena M.

    2016-01-01

    Interconnecting pathways through porous tissue engineering scaffolds play a vital role in determining nutrient supply, cell invasion, and tissue ingrowth. However, the global use of the term “interconnectivity” often fails to describe the transport characteristics of these pathways, giving no clear indication of their potential to support tissue synthesis. This article uses new experimental data to provide a critical analysis of reported methods for the description of scaffold transport pathways, ranging from qualitative image analysis to thorough structural parameterization using X-ray Micro-Computed Tomography. In the collagen scaffolds tested in this study, it was found that the proportion of pore space perceived to be accessible dramatically changed depending on the chosen method of analysis. Measurements of % interconnectivity as defined in this manner varied as a function of direction and connection size, and also showed a dependence on measurement length scale. As an alternative, a method for transport pathway parameterization was investigated, using percolation theory to calculate the diameter of the largest sphere that can travel to infinite distance through a scaffold in a specified direction. As proof of principle, this approach was used to investigate the invasion behavior of primary fibroblasts in response to independent changes in pore wall alignment and pore space accessibility, parameterized using the percolation diameter. The result was that both properties played a distinct role in determining fibroblast invasion efficiency. This example therefore demonstrates the potential of the percolation diameter as a method of transport pathway parameterization, to provide key structural criteria for application-based scaffold design. PMID:26888449

  17. Scaffold-free Three-dimensional Graft From Autologous Adipose-derived Stem Cells for Large Bone Defect Reconstruction

    Science.gov (United States)

    Dufrane, Denis; Docquier, Pierre-Louis; Delloye, Christian; Poirel, Hélène A.; André, Wivine; Aouassar, Najima

    2015-01-01

    Abstract Long bone nonunion in the context of congenital pseudarthrosis or carcinologic resection (with intercalary bone allograft implantation) is one of the most challenging pathologies in pediatric orthopedics. Autologous cancellous bone remains the gold standard in this context of long bone nonunion reconstruction, but with several clinical limitations. We then assessed the feasibility and safety of human autologous scaffold-free osteogenic 3-dimensional (3D) graft (derived from autologous adipose-derived stem cells [ASCs]) to cure a bone nonunion in extreme clinical and pathophysiological conditions. Human ASCs (obtained from subcutaneous adipose tissue of 6 patients and expanded up to passage 4) were incubated in osteogenic media and supplemented with demineralized bone matrix to obtain the scaffold-free 3D osteogenic structure as confirmed in vitro by histomorphometry for osteogenesis and mineralization. The 3D “bone-like” structure was finally transplanted for 3 patients with bone tumor and 3 patients with bone pseudarthrosis (2 congenital, 1 acquired) to assess the clinical feasibility, safety, and efficacy. Although minor clones with structural aberrations (aneuploidies, such as tri or tetraploidies or clonal trisomy 7 in 6%–20% of cells) were detected in the undifferentiated ASCs at passage 4, the osteogenic differentiation significantly reduced these clonal anomalies. The final osteogenic product was stable, did not rupture with forceps manipulation, did not induce donor site morbidity, and was easily implanted directly into the bone defect. No acute (development, were associated with the graft up to 4 years after transplantation. We report for the first time that autologous ASC can be fully differentiated into a 3D osteogenic-like implant without any scaffold. We demonstrated that this engineered tissue can safely promote osteogenesis in extreme conditions of bone nonunions with minor donor site morbidity and no oncological side effects. PMID

  18. 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds.

    Directory of Open Access Journals (Sweden)

    Hang Li

    Full Text Available Neural stem/progenitor cells (NSPCs are the stem cell of the adult central nervous system (CNS. These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes, thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm(2, 10 wt%, 6330±1160 µm(2, 20 wt%, 7600±1550 µm(2 compared with controls (0 wt%, 3150±220 µm(2. Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.

  19. Biomimetic scaffolds containing nanofibers coated with willemite nanoparticles for improvement of stem cell osteogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ramezanifard, Rouhallah [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Seyedjafari, Ehsan, E-mail: seyedjafari@ut.ac.ir [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Ardeshirylajimi, Abdolreza [Department of Stem Cell Biology, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Soleimani, Masoud [Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2016-05-01

    Nowadays, discovering osteogenesis stimulating effectors is one of the major topics in bone tissue engineering and regenerative medicine. In this study, the proliferation rate and osteogenic differentiation potency of adipose-derived mesenchymal stem cells (AT-MSCs) cultured on poly (L-lactide acid) (PLLA) and willemite-coated PLLA were investigated by MTT assay and common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition and bone-related genes expression. Willemite-coated PLLA showed a higher proliferation support to AT-MSCs in comparison to PLLA and TCPS. During the period of study, AT-MSCs cultured on willemite-coated PLLA scaffolds exhibited the greatest ALP activity and mineralization. Gene expression analysis demonstrated that the highest expression of four important osteogenic-related genes, osteonectin, Runx2, collagen type 1 and osteocalcin was observed in stem cells cultured on willemite-coated PLLA nanofibrous scaffolds. According to the results, willemite-coated PLLA could be a suitable substrate to support the proliferation and osteogenic differentiation of stem cells and holds promising potential for bone tissue engineering and regenerative medicine applications. - Highlights: • Biodegradable PLLA eletrospun nanofibrous scaffold was prepared. • PLLA nanofibers were treated with plasma and coated with willemite nanoparticles. • MSC on willemite-coated PLLA showed greater osteogenic differentiation than those on uncoated PLLA and TCPS. • Willemite-coated nanofibers hold promising potential for bone tissue engineering application.

  20. Repair of injured spinal cord using biomaterial scaffolds and stem cells.

    Science.gov (United States)

    Shrestha, Bikesh; Coykendall, Katherine; Li, Yongchao; Moon, Alex; Priyadarshani, Priyanka; Yao, Li

    2014-08-01

    The loss of neurons and degeneration of axons after spinal cord injury result in the loss of sensory and motor functions. A bridging biomaterial construct that allows the axons to grow through has been investigated for the repair of injured spinal cord. Due to the hostility of the microenvironment in the lesion, multiple conditions need to be fulfilled to achieve improved functional recovery. A scaffold has been applied to bridge the gap of the lesion as contact guidance for axonal growth and to act as a vehicle to deliver stem cells in order to modify the microenvironment. Stem cells may improve functional recovery of the injured spinal cord by providing trophic support or directly replacing neurons and their support cells. Neural stem cells and mesenchymal stem cells have been seeded into biomaterial scaffolds and investigated for spinal cord regeneration. Both natural and synthetic biomaterials have increased stem cell survival in vivo by providing the cells with a controlled microenvironment in which cell growth and differentiation are facilitated. This optimal multi‒disciplinary approach of combining biomaterials, stem cells, and biomolecules offers a promising treatment for the injured spinal cord.

  1. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  2. Peptide-Tethered Hydrogel Scaffold Promotes Recovery from Spinal Cord Transection via Synergism with Mesenchymal Stem Cells.

    Science.gov (United States)

    Li, Li-Ming; Han, Min; Jiang, Xin-Chi; Yin, Xian-Zhen; Chen, Fu; Zhang, Tian-Yuan; Ren, Hao; Zhang, Ji-Wen; Hou, Ting-Jun; Chen, Zhong; Ou-Yang, Hong-Wei; Tabata, Yasuhiko; Shen, You-Qing; Gao, Jian-Qing

    2017-02-01

    Spinal cord injury (SCI) is one of the most devastating injuries. Treatment strategies for SCI are required to overcome comprehensive issues. Implantation of biomaterial scaffolds and stem cells has been demonstrated to be a promising strategy. However, a comprehensive recovery effect is difficult to achieve. In the comprehensive treatment process, the specific roles of the implanted scaffolds and of stem cells in combined strategy are usually neglected. In this study, a peptide-modified scaffold is developed based on hyaluronic acid and an adhesive peptide PPFLMLLKGSTR. Synchrotron radiation micro computed tomography measurement provides insights to the three-dimensional inner topographical property and perspective porous structure of the scaffold. The modified scaffold significantly improves cellular survival and adhesive growth of mesenchymal stem cells during 3D culture in vitro. After implantation in transected spinal cord, the modified scaffold and mesenchymal stems are found to function in synergy to restore injured spinal cord tissue, with respective strengths. Hindlimb motor function scores exhibit the most significant impact of the composite implant at 2 weeks post injury, which is the time secondary injury factors begin to take hold. Investigation on the secondary injury factors including inflammatory response and astrocyte overactivity at 10 days post injury reveals the possible underlying reason. Implants of the scaffold, cells, and especially the combination of both elicit inhibitory effects on these adverse factors. The study develops a promising implant for spinal cord tissue engineering and reveals the roles of the scaffold and stem cells. More importantly, the results provide the first understanding of the bioactive peptide PPFLMLLKGSTR concerning its functions on mesenchymal stem cells and spinal cord tissue restoration.

  3. Naturally Occurring Extracellular Matrix Scaffolds for Dermal Regeneration: Do They Really Need Cells?

    Directory of Open Access Journals (Sweden)

    A. M. Eweida

    2015-01-01

    Full Text Available The pronounced effect of extracellular matrix (ECM scaffolds in supporting tissue regeneration is related mainly to their maintained 3D structure and their bioactive components. These decellularized matrix scaffolds could be revitalized before grafting via adding stem cells, fibroblasts, or keratinocytes to promote wound healing. We reviewed the online published literature in the last five years for the studies that performed ECM revitalization and discussed the results of these studies and the related literature. Eighteen articles met the search criteria. Twelve studies included adding cells to acellular dermal matrix (ADM, 3 studies were on small intestinal mucosa (SIS, one study was on urinary bladder matrix (UBM, one study was on amniotic membrane, and one study included both SIS and ADM loaded constructs. We believe that, in chronic and difficult-to-heal wounds, revitalizing the ECM scaffolds would be beneficial to overcome the defective host tissue interaction. This belief still has to be verified by high quality randomised clinical trials, which are still lacking in literature.

  4. Modified bacterial cellulose scaffolds for localized doxorubicin release in human colorectal HT-29 cells.

    Science.gov (United States)

    Cacicedo, Maximiliano L; León, Ignacio E; Gonzalez, Jimena S; Porto, Luismar M; Alvarez, Vera A; Castro, Guillermo R

    2016-04-01

    Bacterial cellulose (BC) films modified by the in situ method with the addition of alginate (Alg) during the microbial cultivation of Gluconacetobacter hansenii under static conditions increased the loading of doxorubicin by at least three times. Biophysical analysis of BC-Alg films by scanning electron microscopy, thermogravimetry, X-ray diffraction and FTIR showed a highly homogeneous interpenetrated network scaffold without changes in the BC crystalline structure but with an increased amorphous phase. The main molecular interactions determined by FTIR between both biopolymers clearly suggest high compatibility. These results indicate that alginate plays a key role in the biophysical properties of the hybrid BC matrix. BC-Alg scaffold analysis by nitrogen adsorption isotherms revealed by the Brunauer-Emmett-Teller (BET) method an increase in surface area of about 84% and in pore volume of more than 200%. The Barrett-Joyner-Halenda (BJH) model also showed an increase of about 25% in the pore size compared to the BC film. Loading BC-Alg scaffolds with different amounts of doxorubicin decreased the cell viability of HT-29 human colorectal adenocarcinoma cell line compared to the free Dox from around 95-53% after 24h and from 63% to 37% after 48 h. Dox kinetic release from the BC-Alg nanocomposite displayed hyperbolic curves related to the different amounts of drug payload and was stable for at least 14 days. The results of the BC-Alg nanocomposites show a promissory potential for anticancer therapies of solid tumors.

  5. Collagen-GAG Scaffolds Grafted Onto Myocardial Infarcts in a Rat Model:A Delivery Vehicle for Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Z.; XIANG; R.; LIAO; M.; KELLY; M.; SPECTOR

    2005-01-01

    1 IntroductionThe objective of the present study was to investigate the response of rat myocardial scar tissue to type I collagen-glycosaminoglycan (GAG) tissue engineering scaffolds, and to assess the feasibility of using a collagen-GAG scaffold as a delivery vehicle for bone marrow-derived mesenchymal stem cells. The benefits of employing the collagen-GAG scaffold for this application include the following:(1) the large surface area of the three-dimensional sponge-like material allows for the delivery of ...

  6. Resorbable polymeric scaffolds for bone tissue engineering: the influence of their microstructure on the growth of human osteoblast-like MG 63 cells.

    Science.gov (United States)

    Pamula, Elzbieta; Filová, Elena; Bacáková, Lucie; Lisá, Vera; Adamczyk, Daniel

    2009-05-01

    Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.

  7. Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering

    Directory of Open Access Journals (Sweden)

    C Lalande

    2011-04-01

    Full Text Available For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide. USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

  8. Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold

    Institute of Scientific and Technical Information of China (English)

    Fei Huang; Qiang Shen; Jitong Zhao

    2013-01-01

    Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study, rat neural stem cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2',3'-cyclic nucleotide 3'-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells.

  9. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue.

    Science.gov (United States)

    Kim, Yong Bok; Lee, Hyeongjin; Yang, Gi-Hoon; Choi, Chang Hyun; Lee, DaeWeon; Hwang, Heon; Jung, Won-Kyo; Yoon, Hyeon; Kim, Geun Hyung

    2016-01-01

    Cell-printing technology has provided a new paradigm for biofabrication, with potential to overcome several shortcomings of conventional scaffold-based tissue regeneration strategies via controlled delivery of various cell types in well-defined target regions. Here we describe a cell-printing method to obtain mechanically reinforced multi-layered cell-embedded scaffolds, formed of micron-scale poly(ε-caprolactone) (PCL)/alginate struts coated with alginate-based bioink. To compare the physical and cellular activities, we used a scaffold composed of pure alginate (without cells) coated PCL/alginate struts as a control. We systematically varied the ratio of alginate cross-linking agent, and determined the optimal cell-coating conditions to form the PCL/alginate struts. Following fabrication of the cell (MG63)-laden PCL/alginate scaffold, the bioactivity was evaluated in vitro. The laden cells exhibited a substantially more developed cytoskeleton compared with those on a control scaffold consisting of the same material composition. Based on these results, the printed cells exhibited a significantly more homogenous distribution within the scaffold compared with the control. Cell proliferation was determined via MTT assays at 1, 3, 7, and 14 days of culture, and the proliferation of the cell-printed scaffold was substantially in excess (∼2.4-fold) of that on the control. Furthermore, the osteogenic activity such as ALP was measured, and the cell-laden scaffold exhibited significantly greater activity (∼3.2-fold) compared with the control scaffold.

  10. Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds.

    Science.gov (United States)

    Fisher, Matthew B; Henning, Elizabeth A; Söegaard, Nicole; Bostrom, Marc; Esterhai, John L; Mauck, Robert L

    2015-06-01

    Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

  11. Fabrication of poly (ϵ-caprolactone) microfiber scaffolds with varying topography and mechanical properties for stem cell-based tissue engineering applications.

    Science.gov (United States)

    Ko, Junghyuk; Mohtaram, Nima Khadem; Ahmed, Farid; Montgomery, Amy; Carlson, Michael; Lee, Patrick C D; Willerth, Stephanie M; Jun, Martin B G

    2014-01-01

    Highly porous poly (ϵ-caprolactone) microfiber scaffolds can be fabricated using electrospinning for tissue engineering applications. Melt electrospinning produces such scaffolds by direct deposition of a polymer melt instead of dissolving the polymer in a solvent as performed during solution electrospinning. The objective of this study was to investigate the significant parameters associated with the melt electrospinning process that influence fiber diameter and scaffold morphology, including processing temperature, collection distance, applied, voltage and nozzle size. The mechanical properties of these microfiber scaffolds varied with microfiber diameter. Additionally, the porosity of scaffolds was determined by combining experimental data with mathematical modeling. To test the cytocompatability of these fibrous scaffolds, we seeded neural progenitors derived from murine R1 embryonic stem cell lines onto these scaffolds, where they could survive, migrate, and differentiate into neurons; demonstrating the potential of these melt electrospun scaffolds for tissue engineering applications.

  12. Making the Failure More Productive: Scaffolding the Invention Process to Improve Inquiry Behaviors and Outcomes in Invention Activities

    Science.gov (United States)

    Holmes, N. G.; Day, James; Park, Anthony H. K.; Bonn, D. A.; Roll, Ido

    2014-01-01

    Invention activities are Productive Failure activities in which students attempt (and often fail) to invent methods that capture deep properties of a construct before being taught expert solutions. The current study evaluates the effect of scaffolding on the invention processes and outcomes, given that students are not expected to succeed in their…

  13. Functionally graded beta-TCP/PCL nanocomposite scaffolds: in vitro evaluation with human fetal osteoblast cells for bone tissue engineering.

    Science.gov (United States)

    Ozkan, Seher; Kalyon, Dilhan M; Yu, Xiaojun

    2010-03-01

    The engineering of biomimetic tissue relies on the ability to develop biodegradable scaffolds with functionally graded physical and chemical properties. In this study, a twin-screw-extrusion/spiral winding (TSESW) process was developed to enable the radial grading of porous scaffolds (discrete and continuous gradations) that were composed of polycaprolactone (PCL), beta-tricalciumphosphate (beta-TCP) nanoparticles, and salt porogens. Scaffolds with interconnected porosity, exhibiting myriad radial porosity, pore-size distributions, and beta-TCP nanoparticle concentration could be obtained. The results of the characterization of their compressive properties and in vitro cell proliferation studies using human fetal osteoblast cells suggest the promising nature of such scaffolds. The significant degree of freedom offered by the TSESW process should be an additional enabler in the quest toward the mimicry of the complex elegance of the native tissues.

  14. Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing.

    Science.gov (United States)

    Moisenovich, M M; Arkhipova, A Yu; Orlova, A A; Drutskaya, M S; Volkova, S V; Zacharov, S E; Agapov, I I; Kirpichnikov, M P

    2014-01-01

    Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems.

  15. Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications.

    Science.gov (United States)

    Coimbra, P; Ferreira, P; de Sousa, H C; Batista, P; Rodrigues, M A; Correia, I J; Gil, M H

    2011-01-01

    In this work, porous scaffolds obtained from the freeze-drying of pectin/chitosan polyelectrolyte complexes were prepared and characterized by FTIR, SEM and weight loss studies. Additionally, the cytotoxicity of the prepared scaffolds was evaluated in vitro, using human osteoblast cells. The results obtained showed that cells adhered to scaffolds and proliferated. The study also confirmed that the degradation by-products of pectin/chitosan scaffold are noncytotoxic.

  16. Tumor cell culture on collagen–chitosan scaffolds as three-dimensional tumor model: A suitable model for tumor studies

    Directory of Open Access Journals (Sweden)

    Aziz Mahmoudzadeh

    2016-07-01

    Full Text Available Tumor cells naturally live in three-dimensional (3D microenvironments, while common laboratory tests and evaluations are done in two-dimensional (2D plates. This study examined the impact of cultured 4T1 cancer cells in a 3D collagen–chitosan scaffold compared with 2D plate cultures. Collagen–chitosan scaffolds were provided and passed confirmatory tests. 4T1 tumor cells were cultured on scaffolds and then tumor cells growth rate, resistance to X-ray radiation, and cyclophosphamide as a chemotherapy drug were analyzed. Furthermore, 4T1 cells were extracted from the scaffold model and were injected into the mice. Tumor growth rate, survival rate, and systemic immune responses were evaluated. Our results showed that 4T1 cells infiltrated the scaffolds pores and constructed a 3D microenvironment. Furthermore, 3D cultured tumor cells showed a slower proliferation rate, increased levels of survival to the X-ray irradiation, and enhanced resistance to chemotherapy drugs in comparison with 2D plate cultures. Transfer of extracted cells to the mice caused enhanced tumor volume and decreased life span. This study indicated that collagen–chitosan nanoscaffolds provide a suitable model of tumor that would be appropriate for tumor studies.

  17. Behaviour of Human Induced Pluripotent Stem Cell-Derived Neural Progenitors on Collagen Scaffolds Varied in Freezing Temperature and Laminin Concentration

    Directory of Open Access Journals (Sweden)

    Fahimeh Khayyatan

    2014-03-01

    Full Text Available Objective: Biomaterial technology, when combined with emerging human induced pluripotent stem cell (hiPSC technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors (hiPSC-NPs. In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. Materials and Methods: Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. Results: Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 μm for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. Conclusion: The results have suggested that the pore structure and laminin coating of

  18. Enhanced cellular activities of polycaprolactone/alginate-based cell-laden hierarchical scaffolds for hard tissue engineering applications.

    Science.gov (United States)

    Lee, HyeongJin; Kim, GeunHyung

    2014-09-15

    Biomedical scaffolds have been widely investigated because they are essential for support and promotion of cell adhesion, proliferation and differentiation in three-dimensional (3D) structures. An ideal scaffold should be highly porous to enable efficient nutrient and oxygen transfer and have a 3D structure that provides optimal micro-environmental conditions for the seeded cells to obtain homogeneous growth after a long culture period. In this study, new hierarchical osteoblast-like cell (MG-63)-laden scaffolds consisting of micro-sized struts/inter-layered micro-nanofibres and cell-laden hydrogel struts with mechanically stable and biologically superior properties were introduced. Poly(ethylene oxide) (PEO) was used as a sacrificial component to generate pores within the cell-laden hydrogel struts to attain a homogeneous cell distribution and rapid cell growth in the scaffold interior. The alginate-based cell-laden struts with PEO induced fast/homogeneous cell release, in contrast to nonporous cell-laden struts. Various weight fractions (0.5, 1, 2, 3 and 3.5 wt%) of PEO were used, of which 2 wt% PEO in the cell-laden strut resulted in the most appropriate cell release and enhanced biological activities (cell proliferation and calcium deposition), compared to nonporous cell-laden struts.

  19. Development of a bone reconstruction technique using a solid free-form fabrication (SFF)-based drug releasing scaffold and adipose-derived stem cells.

    Science.gov (United States)

    Lee, Jin Woo; Kim, Ki-Joo; Kang, Kyung Shin; Chen, Shaochen; Rhie, Jong-Won; Cho, Dong-Woo

    2013-07-01

    For tissue regeneration, three essential components of scaffolds, signals (biomolecules), and cells are required. Moreover, because bony defects are three-dimensional in many clinical circumstances, an exact 3D scaffold is important. Therefore, we proposed an effective reconstruction tool for cranial defects using human adipose-derived stem cells (hADSCs) and a 3D functional scaffold fabricated by solid free-form fabrication (SFF) technology that secretes biomolecules. We fabricated poly(propylene fumarate)-based 3D scaffolds with embedded microsphere-deliverable bone morphogenetic protein-2 (BMP-2) by microstereolithography. BMP-2-loaded SFF scaffolds with/without hADSCs (SFF/BMP/hADSCs scaffolds and SFF/BMP scaffolds, respectively) and BMP-2-unloaded SFF scaffolds (SFF scaffolds) were then implanted in rat crania, and in vivo bone formation was observed. Analyses of bone formation areas using micro-computed tomography (micro-CT) showed the superiority of SFF/BMP/hADSCs scaffolds. Hematoxylin and eosin stain, Masson's trichrome stain, and collagen type-I stain supported the results of the micro-CT scan. And human leukocyte antigen-ABC showed that seeded, differentiated hADSCs were well grown and changed to the bone tissue at the inside of the scaffold. Results showed that our combination of a functional 3D scaffold and hADSCs may be a useful tool for improving the reconstruction quality of severe bony defects in which thick bone is required.

  20. Chitosan-gelatin scaffolds for tissue engineering: physico-chemical properties and biological response of buffalo embryonic stem cells and transfectant of GFP-buffalo embryonic stem cells.

    Science.gov (United States)

    Thein-Han, W W; Saikhun, J; Pholpramoo, C; Misra, R D K; Kitiyanant, Y

    2009-11-01

    The favorable cellular response of newly developed cell line, buffalo embryonic stem (ES) cells to three-dimensional biodegradable chitosan-gelatin composite scaffolds with regard to stem-cell-based tissue engineering is described. Chitosan-gelatin composites were characterized by a highly porous structure with interconnected pores, and the mechanical properties were significantly enhanced. Furthermore, X-ray diffraction study indicated increased amorphous content in the scaffold on the addition of gelatin to chitosan. To develop a transfectant of green fluorescence protein (GFP)-buffalo ES cell, transfection of GFP plasmid to the cell was carried out via the electroporation procedure. In comparison with pure chitosan, cell spreading and proliferation were greater in highly visualized GFP-expressing cell-chitosan-gelatin scaffold constructs. The relative comparison of biological response involving cell proliferation and viability on the scaffolds suggests that blending of gelatin in chitosan improved cellular efficiency. Studies involving scanning electron and fluorescence microscopy, histological observations and flow cytometer analysis of the constructs implied that the polygonal cells attached to and penetrated the pores, and proliferated well, while maintaining their pluripotency during the culture period for 28days. Chitosan-gelatin scaffolds were cytocompatible with respect to buffalo ES cells. The study underscores for the first time that chitosan-gelatin scaffolds are promising candidates for ES-cell-based tissue engineering.

  1. Polyhydroxyalkanoates: waste glycerol upgrade into electrospun fibrous scaffolds for stem cells culture.

    Science.gov (United States)

    Canadas, Raphaël F; Cavalheiro, João M B T; Guerreiro, João D T; de Almeida, M Catarina M D; Pollet, Eric; da Silva, Cláudia Lobato; da Fonseca, M M R; Ferreira, Frederico Castelo

    2014-11-01

    This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic the structure of the native extracellular matrix, the natural environment where cells inhabit. Following a biorefinery approach, crude glycerol directly recovered from a biodiesel post-reaction stream was fed as major C source to Cupriavidus necator DSM 545 to produce polyhydroxyalkanoates at polymer titers of 9-25g/L. Two of the P(3HB-4HB-3HV) terpolymers produced, one containing 11.4% 4HB and 3.5% 3HV and the other containing 35.6% 4HB and 3.4% 3HV, were electrospun into fibers of average diameters of 600 and 1400nm, respectively. hMSC were cultured for 7 days in both fiber meshes, showing their ability to support stem cell growth at acceptable proliferation levels. Comparative results clearly demonstrate that scaffold topology is critical, with electrospun PHA fibers succeeding on the support of significant cell adhesion and proliferation, where planar PHA films failed.

  2. Cell-laden poly(ɛ-caprolactone)/alginate hybrid scaffolds fabricated by an aerosol cross-linking process for obtaining homogeneous cell distribution: fabrication, seeding efficiency, and cell proliferation and distribution.

    Science.gov (United States)

    Lee, HyeongJin; Ahn, SeungHyun; Bonassar, Lawrence J; Chun, Wook; Kim, GeunHyung

    2013-10-01

    Generally, solid-freeform fabricated scaffolds show a controllable pore structure (pore size, porosity, pore connectivity, and permeability) and mechanical properties by using computer-aided techniques. Although the scaffolds can provide repeated and appropriate pore structures for tissue regeneration, they have a low biological activity, such as low cell-seeding efficiency and nonuniform cell density in the scaffold interior after a long culture period, due to a large pore size and completely open pores. Here we fabricated three different poly(ɛ-caprolactone) (PCL)/alginate scaffolds: (1) a rapid prototyped porous PCL scaffold coated with an alginate, (2) the same PCL scaffold coated with a mixture of alginate and cells, and (3) a multidispensed hybrid PCL/alginate scaffold embedded with cell-laden alginate struts. The three scaffolds had similar micropore structures (pore size = 430-580 μm, porosity = 62%-68%, square pore shape). Preosteoblast cells (MC3T3-E1) were used at the same cell density in each scaffold. By measuring cell-seeding efficiency, cell viability, and cell distribution after various periods of culturing, we sought to determine which scaffold was more appropriate for homogeneously regenerated tissues.

  3. Biocompatibility of various hydoxyapatite scaffolds evaluated by proliferation of rat’s bone marrow mesenchymal stem cells: an in vitro study

    Directory of Open Access Journals (Sweden)

    Achmad F. Kamal

    2013-12-01

    Full Text Available Background: Scaffold (biomaterial biocompatibility test should be performed in vitro prior to in vivo stem cell application in animal or clinical trial. These test consists of direct and indirect toxicity test (MTT assay [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide]. Those tests were used to identify cell morphological changes, cell-substrate adhesion impairment, and reduction in cell proliferation activity.Methods: The tested scaffolds were hydroxyapatite-calcium sulphate (HA-CaSO4 (scaffold I, nano-particular HA paste (scaffold II, synthetic HA granule (scaffold III, bovine HA granule (scaffold IV, and morsellized bovine xenograft (scaffold V. Direct contact toxicity test and MTT assay [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] were performed on those groups. In direct contact toxicity test, we put granules of various scaffolds within plates and incubated together with mesenchymal stem cells (MSCs. In MTT assay we included phenol 20 mg/mL and 100 mg/mL group as positive control. Morphology, cell adhesion impairment, and cell growth were monitored daily until day-7. Cells counting in the direct contact toxicity test was conducted on day-7.Results: There were no changes on 24 hours observation after direct contact. On day-7, an impairment of cell adhesion to plastic substrates, changes in cell morphology, and cell death were observed, especially in scaffold I, scaffold II, and scaffold V. In MTT assay, only scaffold I, phenol 20 mg/mL, and phenol 100 mg/mL showed more than 50% inhibition at 24-hour and 7-day-observation. Extracts from scaffold II, III, IV, and V did not affect the viability and proliferation of bone marrow MSCs (inhibition value < 50%. Scaffold II, III, IV and V were proven non-cytotoxic and have good biocompatibility in vitro,  no statistical significant differences were observed among the scaffold groups (p > 0.05.Conclusion: We understand which scaffold was nontoxic or the least toxic to

  4. Synthesis and Characterization of Carboxymethylcellulose-Methacrylate Hydrogel Cell Scaffolds

    OpenAIRE

    2010-01-01

    Many carbohydrates pose advantages for tissue engineering applications due to their hydrophilicity, degradability, and availability of chemical groups for modification. For example, carboxymethylcellulose (CMC) is a water-soluble cellulose derivative that is degradable by cellulase. Though this enzyme is not synthesized by mammalian cells, cellulase and the fragments derived from CMC degradation are biocompatible. With this in mind, we created biocompatible, selectively degradable CMC-based h...

  5. Conducting cryogel scaffold as a potential biomaterial for cell stimulation and proliferation.

    Science.gov (United States)

    Vishnoi, Tanushree; Kumar, Ashok

    2013-02-01

    The aim of the study was to demonstrate the potential of the cryogelation technique for the synthesis of the conducting cryogel scaffolds which would encompass the advantages of the cryogel matrix, like the mechanical strength and interconnected porous network as well as the conductive properties of the incorporated conducting polymeric material, polypyrrole. The cryogels were synthesized using different combinations of oxidizing agents and surfactants like, sodium dodecyl sulfate (SDS)/ammonium persulfate (APS), SDS/iron chloride (FeCl(3)), cetyl trimethyl ammonium bromide (CTAB)/APS, and CTAB/FeCl(3). The synthesized gels were characterized by scanning electron microscopic analysis for morphology, Fourier transform infrared spectroscopy for analyzing the presence of the polypyrrole (0.5-4 %) as nano-fillers in the gel. It was observed that the presence of these nano-fillers increased the swelling ratio by approximately 50 %. The synthesized conducting cryogels displayed high stress bearing capacity without being deformed as analysed by rheological measurements. The degradation studies showed 12-15 % degradation in 4 weeks time. In vitro studies with conducting and non-conducting cryogel scaffold were carried out to optimize the stimulation conditions for the two cell lines, neuro2a and cardiac muscle C2C12. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed approximately 25 and 15 % increase in the cell proliferation rate for neuro2a and C2C12 cell line, respectively. This was observed at a specific voltage of 100 mV and 2 V, for a specified duration of 2 h and 1 min, respectively for the conducting scaffold as compared to the control. This can play an important role in tissue engineering applications for cell lines where acquiring a high cell number and functionality is desired.

  6. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Aaron X Sun

    2015-08-01

    Full Text Available Poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light based PSL (VL-PSL system to encapsulate human adipose-derived stem cells (hASCs into a biodegradable polymer (poly-D,L-lactic acid/polyethylene glycol/ poly-D,L-lactic acid (PDLLA-PEG/hyaluronic acid (HA matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84% and were uniformly distributed throughout the constructs, which possessed high mechanical property with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in Control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium treated group (TGF-β3 group hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 x 10(5 fold increases, respectively, compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan (GAG-rich extracellular matrix, detected by immunohistochemistry, and Alcian blue and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group, cell viability decreased with time (65% at 28 days and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL- and PLLA-PEG/HA based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage

  7. Use of synovium-derived stromal cells and chitosan/collagen type I scaffolds for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Gong Zhongcheng; Lin Zhaoquan [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054 (China); Xiong Hui; Long Xing; Wei Lili; Li Jian; Wu Yang, E-mail: xinglong1957@yahoo.com.c [State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079 (China)

    2010-10-01

    The objective was to investigate synovium-derived stromal cells (SDSCs) coupled with chitosan/collagen type I (CS/COL-I) scaffolds for cartilage engineering. CS/COL-I scaffolds were fabricated through freeze-drying and cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. SDSCs were isolated from synovium and cultured onto CS/COL-I scaffolds, constructs of which were incubated in serum-free chondrogenic medium with sequential application of TGF-{beta}1 and bFGF for up to 21 days and then implanted into nude mice. The physical characteristics of the scaffolds were examined. The quality of the in vitro constructs was assessed in terms of DNA content by PicoGreen assay and cartilaginous matrix by histological examination. The implants of the constructs were evaluated by histological and immunohistochemical examinations and reverse transcription PCR. Results indicated that the CS/COL-I scaffold showed porous structures, and the DNA content of SDSCs in CS/COL-I scaffolds increased at 1 week culture time. Both of the constructs in vitro and the implants were examined with positive stained GAGs histologically and the implants with positive collagen type II immunohistochemically. RT-PCR of the implants indicated that aggrecan and collagen type II expressed. It suggested that SDSCs coupled with CS/COL-I scaffolds treated sequentially with TGF-{beta}1 and bFGF in vitro were highly competent for engineered cartilage formation in vitro and in vivo.

  8. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Clément Achille

    Full Text Available RNA interference (RNAi is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL alone (Control and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i and EGFP (EGFPi, also served as a control shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing and 0.2-3 µm (Control. While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

  9. The fabrication of double layer tubular vascular tissue engineering scaffold via coaxial electrospinning and its 3D cell coculture.

    Science.gov (United States)

    Ye, Lin; Cao, Jie; Chen, Lamei; Geng, Xue; Zhang, Ai-Ying; Guo, Lian-Rui; Gu, Yong-Quan; Feng, Zeng-Guo

    2015-12-01

    A continuous electrospinning technique was applied to fabricate double layer tubular tissue engineering vascular graft (TEVG) scaffold. The luminal layer was made from poly(ɛ-caprolac-tone)(PCL) ultrafine fibers via common single axial electrospinning followed by the outer layer of core-shell structured nanofibers via coaxial electrospinning. For preparing the outer layernano-fibers, the PCL was electrospun into the shell and both bovine serum albumin (BSA) and tetrapeptide val-gal-pro-gly (VAPG) were encapsulated into the core. The core-shell structure in the outer layer fibers was observed by transmission electron microscope (TEM). The in vitro release tests exhibited the sustainable release behavior of BSA and VAPG so that they provided a better cell growth environment in the interior of tubular scaffold wall. The in vitro culture of smooth muscle cells (SMCs) demonstrated their potential to penetrate into the scaffold wall for the 3D cell culture. Subsequently, 3D cell coculture was conducted. First, SMCs were seeded on the luminal surface of the scaffold and cultured for 5 days, and then endothelial cells (ECs) were also seeded on the luminal surface and cocultured with SMCs for another 2 days. After stained with antibodies, 3D cell distribution on the scaffold was revealed by confocal laser scanning microscopy (CLSM) where ECs were mainly located on the luminal surface whereas SMCs penetrated into the surface and distributed inside the scaffold wall. This double layer tubular scaffold with 3D cell distribution showed the promise to develop it into a novel TEVG for clinical trials in the near future.

  10. In vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures.

    Science.gov (United States)

    Abdel-Rahman, Iman A M; Beuerle, Till; Ernst, Ludger; Abdel-Baky, Afaf M; Desoky, Ezz El-Din K; Ahmed, Amany S; Beerhues, Ludger

    2013-04-01

    The anthranoid skeleton is believed to be formed by octaketide synthase (OKS), a member of the type III polyketide synthase (PKS) superfamily. Recombinant OKSs catalyze stepwise condensation of eight acetyl units to form a linear octaketide intermediate which, however, is incorrectly folded and cyclized to give the shunt products SEK4 and SEK4b. Here we report in vitro formation of the anthranoid scaffold by cell-free extracts from yeast-extract-treated Cassia bicapsularis cell cultures. Unlike field- and in vitro-grown shoots which accumulate anthraquinones, cell cultures mainly contained tetrahydroanthracenes, formation of which was increased 2.5-fold by the addition of yeast extract. The elicitor-stimulated accumulation of tetrahydroanthracenes was preceded by an approx. 35-fold increase in OKS activity. Incubation of cell-free extracts from yeast-extract-treated cell cultures with acetyl-CoA and [2-(14)C]malonyl-CoA led to formation of torosachrysone (tetrahydroanthracene) and emodin anthrone, beside two yet unidentified products. No product formation occurred in the absence of acetyl-CoA as starter substrate. To confirm the identities of the enzymatic products, cell-free extracts were incubated with acetyl-CoA and [U-(13)C(3)]malonyl-CoA and (13)C incorporation was analyzed by ESI-MS/MS. Detection of anthranoid biosynthesis in cell-free extracts indicates in vitro cooperation of OKS with a yet unidentified factor or enzyme for octaketide cyclization.

  11. Gas-foaming calcium phosphate cement scaffold encapsulating human umbilical cord stem cells.

    Science.gov (United States)

    Chen, Wenchuan; Zhou, Hongzhi; Tang, Minghui; Weir, Michael D; Bao, Chongyun; Xu, Hockin H K

    2012-04-01

    Tissue engineering approaches are promising to meet the increasing need for bone regeneration. Calcium phosphate cement (CPC) can be injected and self-set to form a scaffold with excellent osteoconductivity. The objectives of this study were to develop a macroporous CPC-chitosan-fiber construct containing alginate-fibrin microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) and to investigate hUCMSC release from the degrading microbeads and proliferation inside the porous CPC construct. The hUCMSC-encapsulated microbeads were completely wrapped inside the CPC paste, with the gas-foaming porogen creating macropores in CPC to provide for access to culture media. Increasing the porogen content in CPC significantly increased the cell viability, from 49% of live cells in CPC with 0% porogen to 86% of live cells in CPC with 15% porogen. The alginate-fibrin microbeads started to degrade and release the cells inside CPC at 7 days. The released cells started to proliferate inside the macroporous CPC construct. The live cell number inside CPC increased from 270 cells/mm(2) at 1 day to 350 cells/mm(2) at 21 days. The pore volume fraction of CPC increased from 46.8% to 78.4% using the gas-foaming method, with macropore sizes of approximately 100 to 400 μm. The strength of the CPC-chitosan-fiber scaffold at 15% porogen was 3.8 MPa, which approximated the reported 3.5 MPa for cancellous bone. In conclusion, a novel gas-foaming macroporous CPC construct containing degradable alginate-fibrin microbeads was developed that encapsulated hUCMSCs. The cells had good viability while wrapped inside the porous CPC construct. The degradable microbeads in CPC quickly released the cells, which proliferated over time inside the porous CPC. Self-setting, strong CPC with alginate-fibrin microbeads for stem cell delivery is promising for bone tissue engineering applications.

  12. Tuning of perovskite solar cell performance via low-temperature brookite scaffolds surface modifications

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    Trilok Singh

    2017-01-01

    Full Text Available The nature of metal oxide scaffold played a pivotal role for the growth of high quality perovskites and subsequently facilitates efficient photovoltaics devices. We demonstrate an effective way to fabricate a low-temperature TiO2 brookite scaffold layer with a uniform and pinhole-free layer for enhancing photovoltaic properties of perovskite solar cells. Various concentrations of TiCl4 were used to modify brookite TiO2 for efficient charge generation and fast charge extraction. We observed that the brookite layer with an appropriate TiCl4 treatment possesses a smooth surface with full coverage of the substrates, whereas TiCl4 treatment further improves the contact of the TiO2/perovskite interface which facilitates charge extraction and drastically influenced charge recombination. The surface treated brookite scaffolds perovskite devices showed an improved performance with an average power conversion efficiency more than 17%. The time resolved photoluminescence showed that the treated samples have obvious effect on the charge carrier dynamics. The striking observation of this study was very low appearance of hysteresis and high reproducibility in the treated samples, which opens up the possibilities for the fabrication of high efficient devices at relatively low temperatures with negligible hysteresis via facile surface modifications.

  13. Effects of Ciprofloxacin-Containing Antimicrobial Scaffolds on Dental Pulp Stem Cell Viability — In Vitro Studies

    Science.gov (United States)

    Kamocki, Krzysztof; Nör, Jacques E.; Bottino, Marco C.

    2017-01-01

    Objective A combination of antibiotics, including but not limited to metronidazole (MET) and ciprofloxacin (CIP), has been indicated to eradicate bacteria in necrotic immature permanent teeth prior to regenerative procedures. It has been shown clinically that antibiotic pastes may lead to substantial stem cell death. The aim of this study was to synthesize scaffolds containing various concentrations of CIP to enhance cell viability while preserving antimicrobial properties. Design Polydioxanone (PDS)-based electrospun scaffolds were processed with decreasing CIP concentrations (25 – 1 wt.%) and morphologically evaluated using scanning electron microscopy (SEM). Cytotoxicity assays were performed to determine whether the amount of CIP released from the scaffolds would lead to human dental pulp stem cell (hDPSC) toxicity. Similarly, WST-1 assays were performed to evaluate the impact of CIP release on hDPSC proliferation. Pure PDS scaffolds and saturated double antibiotic solution MET/CIP (DAP) served as both positive and negative controls, respectively. Antibacterial efficacy against E. faecalis (Ef) was tested. Results A significant decrease in hDPSC’ viability at concentrations 5–25 wt.% was observed. However, concentrations below 5 wt.% did not impair cell viability. Data from the WST-1 assays indicated no detrimental impact on cell proliferation for scaffolds containing 2.5 wt.% CIP or less. Significant antimicrobial properties were seen for CIP-scaffolds at lower concentrations (i.e., 1 and 2.5 wt.%). Conclusion The obtained data demonstrated that a reduced concentration of CIP incorporated into PDS-based scaffolds maintains its antimicrobial properties while enhancing viability and proliferation of dental pulp stem cells. PMID:26042622

  14. Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    A Phadke

    2013-01-01

    Full Text Available Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue regeneration and repair. In this study, we investigated the effect of the microarchitecture of the scaffold on osteogenic differentiation of human mesenchymal stem cells (hMSCs. Poly(ethylene glycol diacrylate-co-N-acryloyl 6-aminocaproic acid cryogels were fabricated to have either a pore network consisting of cellular, randomly oriented pores (termed ‘spongy’ or a pore network consisting of lamellar columns (termed ‘columnar’, with both cryogel types showing a similar porosity. Both spongy and columnar cryogels supported comparable levels of cell viability and proliferation of hMSCs in vitro. However, spongy cryogels promoted osteogenic differentiation to a greater extent than their columnar counterparts, as evidenced by increased alkaline phosphatase activity and osteoblastic gene expression over 21 days post culture. Leveraging upon our previous work, we further evaluated the ability of these synthetic scaffolds in conjunction with mineralisation to promote ectopic bone formation upon subcutaneous implantation in nude rats. Mineralised spongy and columnar cryogels, both in the presence and absence of exogenous hMSCs, promoted ectopic bone formation in vivo. No such bone formation was observed in acellular cryogels devoid of mineralisation, with extensive host cell infiltration and vascularisation in columnar cryogels, and negligible infiltration into spongy cryogels. Our results thus present a novel method to tune the microarchitecture of porous polymeric scaffolds, in addition to suggesting their efficacy as synthetic bone grafts.

  15. Nanofibrous Chitosan-Polyethylene Oxide Engineered Scaffolds: A Comparative Study between Simulated Structural Characteristics and Cells Viability

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    Mohammad Kazemi Pilehrood

    2014-01-01

    Full Text Available 3D nanofibrous chitosan-polyethylene oxide (PEO scaffolds were fabricated by electrospinning at different processing parameters. The structural characteristics, such as pore size, overall porosity, pore interconnectivity, and scaffold percolative efficiency (SPE, were simulated by a robust image analysis. Mouse fibroblast cells (L929 were cultured in RPMI for 2 days in the presence of various samples of nanofibrous chitosan/PEO scaffolds. Cell attachments and corresponding mean viability were enhanced from 50% to 110% compared to that belonging to a control even at packed morphologies of scaffolds constituted from pores with nanoscale diameter. To elucidate the correlation between structural characteristics within the depth of the scaffolds’ profile and cell viability, a comparative analysis was proposed. This analysis revealed that larger fiber diameters and pore sizes can enhance cell viability. On the contrary, increasing the other structural elements such as overall porosity and interconnectivity due to a simultaneous reduction in fiber diameter and pore size through the electrospinning process can reduce the viability of cells. In addition, it was found that manipulation of the processing parameters in electrospinning can compensate for the effects of packed morphologies of nanofibrous scaffolds and can thus potentially improve the infiltration and viability of cells.

  16. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells

    Science.gov (United States)

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-01

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold

  17. Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold.

    Science.gov (United States)

    Navarra, Giovanna; Peres, Chiara; Contardi, Marco; Picone, Pasquale; San Biagio, Pier Luigi; Di Carlo, Marta; Giacomazza, Daniela; Militello, Valeria

    2016-09-15

    Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM and SEM images to have a picture of their different spatial arrangement. Finally, the use of the BSA hydrogels as scaffold has been tested in two different cell cultures.

  18. ASTM international workshop on standards and measurements for tissue engineering scaffolds.

    Science.gov (United States)

    Simon, Carl G; Yaszemski, Michael J; Ratcliffe, Anthony; Tomlins, Paul; Luginbuehl, Reto; Tesk, John A

    2015-07-01

    The "Workshop on Standards & Measurements for Tissue Engineering Scaffolds" was held on May 21, 2013 in Indianapolis, IN, and was sponsored by the ASTM International (ASTM). The purpose of the workshop was to identify the highest priority items for future standards work for scaffolds used in the development and manufacture of tissue engineered medical products (TEMPs). Eighteen speakers and 78 attendees met to assess current scaffold standards and to prioritize needs for future standards. A key finding was that the ASTM TEMPs subcommittees (F04.41-46) have many active "guide" documents for educational purposes, but few standard "test methods" or "practices." Overwhelmingly, the most clearly identified need was standards for measuring the structure of scaffolds, followed by standards for biological characterization, including in vitro testing, animal models and cell-material interactions. The third most pressing need was to develop standards for assessing the mechanical properties of scaffolds. Additional needs included standards for assessing scaffold degradation, clinical outcomes with scaffolds, effects of sterilization on scaffolds, scaffold composition, and drug release from scaffolds. Discussions highlighted the need for additional scaffold reference materials and the need to use them for measurement traceability. Workshop participants emphasized the need to promote the use of standards in scaffold fabrication, characterization, and commercialization. Finally, participants noted that standards would be more broadly accepted if their impact in the TEMPs community could be quantified. Many scaffold standard needs have been identified and focus is turning to generating these standards to support the use of scaffolds in TEMPs.

  19. TiO2 Sub-microsphere Film as Scaffold Layer for Efficient Perovskite Solar Cells.

    Science.gov (United States)

    Huang, Yang; Zhu, Jun; Ding, Yong; Chen, Shuanghong; Zhang, Changneng; Dai, Songyuan

    2016-03-01

    TiO2 sub-microspheres composed of anatase granular-like nanocrystallines with an average diameter ∼250 nm are synthesized using sol-gel method and employed as the scaffold layer for efficient mesocopic perovskite solar cells. Compared with mesoporous TiO2 films composed of ∼18 nm nanoparticles, the sub-microsphere films show superior light-trapping characteristics and significantly improve the light-harvesting capability of the solar cells. In addition, the charge-transport performance is also dramatically improved according to the transient photocurrent decay despite there being no significant difference in the perovskite layer surface morphology. As a result, an average power conversion efficiency of 15% with a highly uniform distribution is achieved for the solar cells with TiO2 sub-microsphere films, 12% higher than those with TiO2 nanoparticle films. The combination of light-harvesting capability and fast charge transfer make the TiO2 sub-microsphere film a good candidate as the scaffold layer for efficient perovskite solar cells.

  20. Influence of Additive Manufactured Scaffold Architecture on the Distribution of Surface Strains and Fluid Flow Shear Stresses and Expected Osteochondral Cell Differentiation.

    Science.gov (United States)

    Hendrikson, Wim J; Deegan, Anthony J; Yang, Ying; van Blitterswijk, Clemens A; Verdonschot, Nico; Moroni, Lorenzo; Rouwkema, Jeroen

    2017-01-01

    Scaffolds for regenerative medicine applications should instruct cells with the appropriate signals, including biophysical stimuli such as stress and strain, to form the desired tissue. Apart from that, scaffolds, especially for load-bearing applications, should be capable of providing mechanical stability. Since both scaffold strength and stress-strain distributions throughout the scaffold depend on the scaffold's internal architecture, it is important to understand how changes in architecture influence these parameters. In this study, four scaffold designs with different architectures were produced using additive manufacturing. The designs varied in fiber orientation, while fiber diameter, spacing, and layer height remained constant. Based on micro-CT (μCT) scans, finite element models (FEMs) were derived for finite element analysis (FEA) and computational fluid dynamics (CFD). FEA of scaffold compression was validated using μCT scan data of compressed scaffolds. Results of the FEA and CFD showed a significant impact of scaffold architecture on fluid shear stress and mechanical strain distribution. The average fluid shear stress ranged from 3.6 mPa for a 0/90 architecture to 6.8 mPa for a 0/90 offset architecture, and the surface shear strain from 0.0096 for a 0/90 offset architecture to 0.0214 for a 0/90 architecture. This subsequently resulted in variations of the predicted cell differentiation stimulus values on the scaffold surface. Fluid shear stress was mainly influenced by pore shape and size, while mechanical strain distribution depended mainly on the presence or absence of supportive columns in the scaffold architecture. Together, these results corroborate that scaffold architecture can be exploited to design scaffolds with regions that guide specific tissue development under compression and perfusion. In conjunction with optimization of stimulation regimes during bioreactor cultures, scaffold architecture optimization can be used to improve

  1. Preseeding of human vascular cells in decellularized bovine pericardium scaffold for tissue-engineered heart valve : An in vitro and in vivo feasibility study

    NARCIS (Netherlands)

    Yang, Min; Chen, Chang-Zhi; Shu, Yu-Sheng; Shi, Wei-Ping; Cheng, Shao-Fei; Gu, Y. John

    2012-01-01

    Human vascular cells from saphenous veins have been used for cell seeding on the synthetic scaffolds for constructing tissue-engineered heart valve (TEHV). However, little is known about the seeding of human vascular cells on bovine pericardium, a potential natural scaffold for TEHV. This study was

  2. Scaffold-integrated microchips for end-to-end in vitro tumor cell attachment and xenograft formation.

    Science.gov (United States)

    Lee, Jungwoo; Kohl, Nathaniel; Shanbhang, Sachin; Parekkadan, Biju

    2015-12-01

    Microfluidic technologies have substantially advanced cancer research by enabling the isolation of rare circulating tumor cells (CTCs) for diagnostic and prognostic purposes. The characterization of isolated CTCs has been limited due to the difficulty in recovering and growing isolated cells with high fidelity. Here, we present a strategy that uses a 3D scaffold, integrated into a microfludic device, as a transferable substrate that can be readily isolated after device operation for serial use in vivo as a transplanted tissue bed. Hydrogel scaffolds were incorporated into a PDMS fluidic chamber prior to bonding and were rehydrated in the chamber after fluid contact. The hydrogel matrix completely filled the fluid chamber, significantly increasing the surface area to volume ratio, and could be directly visualized under a microscope. Computational modeling defined different flow and pressure regimes that guided the conditions used to operate the chip. As a proof of concept using a model cell line, we confirmed human prostate tumor cell attachment in the microfluidic scaffold chip, retrieval of the scaffold en masse, and serial implantation of the scaffold to a mouse model with preserved xenograft development. With further improvement in capture efficiency, this approach can offer an end-to-end platform for the continuous study of isolated cancer cells from a biological fluid to a xenograft in mice.

  3. Human dental pulp stem cell behavior using natural nanotolith/bacterial cellulose scaffolds for regenerative medicine.

    Science.gov (United States)

    Olyveira, Gabriel Molina; Acasigua, Gerson Arisoly Xavier; Costa, Ligia Maria Manzine; Scher, Cristiane Regina; Xavier Filho, Lauro; Pranke, Patricia Helena Lucas; Basmaji, Pierre

    2013-08-01

    Adhesion and Viability study with human dental pulp stem cell using natural nanotolith/bacterial cellulose scaffolds for regenerative medicine are presented at first time in this work. Nanotolith, are osteoinductors, i.e., they stimulate bone regeneration, enabling higher cells migration for bone tissue regeneration formation. This is mainly because nanotoliths are rich minerals present in the internal ear of bony fish. In addition, are part of a system which acts as a depth sensor and balance, acting as a sound vibrations detector and considered essential for the bone mineralization process, as in hydroxiapatites. Nanotoliths influence in bacterial cellulose was analyzed using transmission infrared spectroscopy (FTIR). Results shows that fermentation process and nanotoliths agglomeration decrease initial human dental pulp stem cell adhesion however tested bionanocomposite behavior has cell viability increase over time.

  4. Novel polymeric scaffolds using protein microbubbles as porogen and growth factor carriers.

    Science.gov (United States)

    Nair, Ashwin; Thevenot, Paul; Dey, Jagannath; Shen, Jinhui; Sun, Man-Wu; Yang, Jian; Tang, Liping

    2010-02-01

    Polymeric tissue engineering scaffolds prepared by conventional techniques like salt leaching and phase separation are greatly limited by their poor biomolecule-delivery abilities. Conventional methods of incorporation of various growth factors, proteins, and/or peptides on or in scaffold materials via different crosslinking and conjugation techniques are often tedious and may affect scaffold's physical, chemical, and mechanical properties. To overcome such deficiencies, a novel two-step porous scaffold fabrication procedure has been created in which bovine serum albumin microbubbles (henceforth MB) were used as porogen and growth factor carriers. Polymer solution mixed with MB was phase separated and then lyophilized to create porous scaffold. MB scaffold triggered substantially lesser inflammatory responses than salt-leached and conventional phase-separated scaffolds in vivo. Most importantly, the same technique was used to produce insulin-like growth factor-1 (IGF-1)-eluting porous scaffolds, simply by incorporating IGF-1-loaded MB (MB-IGF-1) with polymer solution before phase separation. In vitro such MB-IGF-1 scaffolds were able to promote cell growth to a much greater extent than scaffold soaked in IGF-1, confirming the bioactivity of the released IGF-1. Further, such MB-IGF-1 scaffolds elicited IGF-1-specific collagen production in the surrounding tissue in vivo. This novel growth factor-eluting scaffold fabrication procedure can be used to deliver a range of single or combination of bioactive biomolecules to substantially promote cell growth and function in degradable scaffold.

  5. Stromal cell derived factor-1α (SDF-1α) directed chemoattraction of transiently CXCR4 overexpressing mesenchymal stem cells into functionalized three-dimensional biomimetic scaffolds

    DEFF Research Database (Denmark)

    Thieme, S; Ryser, Martin; Gentsch, Marcus

    2009-01-01

    into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute...... scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before...... CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS...

  6. Probing the role of scaffold dimensionality and media composition on matrix production and phenotype of fibroblasts.

    Science.gov (United States)

    Chawla, S; Chameettachal, S; Ghosh, S

    2015-04-01

    Porous sponges, hydrogels, and micro/nanofibrous matrix are most commonly used three dimensional (3D) biomaterials in tissue engineering; however, reciprocal interaction between internal dimensionality of biomaterials and fibroblasts remains largely unexplored. Such studies would have potential to generate valuable insights about wound healing, tissue morphogenesis and homeostasis. To the best of our knowledge this is the first study to evaluate functionality of porous collagen matrix and collagen gels for in vitro culture of fibroblasts while investigating the role of culture media composition in modulating morphology, phenotype, extracellular matrix (ECM)-related gene expression and protein synthesis by fibroblasts. Encapsulation of fibroblasts in collagen gel was found to be more effective for ECM production compared to scaffold-based culture, as evidenced by enhanced collagen type I, elastin, lysyl oxidase, aggrecan gene expression. High glucose media induced spindle like morphology of typical in vivo fibroblasts and enhanced collagen production compared to other media. This variation in biosynthesis in different glucose concentrations was possibly due to endogenous activation of TGF-β or by an increase in ATP consuming anabolic pathways in high glucose concentration.

  7. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release.

    Science.gov (United States)

    Yao, Qingqing; Li, Wei; Yu, Shanshan; Ma, Liwei; Jin, Dayong; Boccaccini, Aldo R; Liu, Yong

    2015-11-01

    Novel chitosan-polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering.

  8. Genipin-crosslinked cartilage-derived matrix as a scaffold for human adipose-derived stem cell chondrogenesis.

    Science.gov (United States)

    Cheng, Nai-Chen; Estes, Bradley T; Young, Tai-Horng; Guilak, Farshid

    2013-02-01

    Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering.

  9. Incorporation of mesoporous silica nanoparticles into random electrospun PLGA and PLGA/gelatin nanofibrous scaffolds enhances mechanical and cell proliferation properties

    Energy Technology Data Exchange (ETDEWEB)

    Mehrasa, Mohammad [Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Asadollahi, Mohammad Ali, E-mail: ma.asadollahi@ast.ui.ac.ir [Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Nasri-Nasrabadi, Bijan [Department of Chemical Engineering, Isfahan University of Technology, Isfahan (Iran, Islamic Republic of); Ghaedi, Kamran [Department of Biology, Faculty of Science, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Salehi, Hossein [Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan (Iran, Islamic Republic of); Dolatshahi-Pirouz, Alireza [DTU Nanotech, Center for Nanomedicine and Theranostics, Technical University of Denmark (DTU), DK-2800 Kgs. Lyngby (Denmark); Arpanaei, Ayyoob, E-mail: arpanaei@yahoo.com [Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of)

    2016-09-01

    Poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin random nanofibrous scaffolds embedded with different amounts of mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. To evaluate the effects of nanoparticles on the scaffolds, physical, chemical, and mechanical properties as well as in vitro degradation behavior of scaffolds were investigated. The mean diameters of nanofibers were 974 ± 68 nm for the pure PLGA scaffolds vs 832 ± 70, 764 ± 80, and 486 ± 64 for the PLGA/gelatin, PLGA/10 wt% MSNPs, and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The results suggested that the incorporation of gelatin and MSNPs into PLGA-based scaffolds enhances the hydrophilicity of scaffolds due to an increase of hydrophilic functional groups on the surface of nanofibers. With porosity examination, it was concluded that the incorporation of MSNPs and gelatin decrease the porosity of scaffolds. Nanoparticles also improved the tensile mechanical properties of scaffolds. Using in vitro degradation analysis, it was shown that the addition of nanoparticles to the nanofibers matrix increases the weight loss percentage of PLGA-based samples, whereas it decreases the weight loss percentage in the PLGA/gelatin composites. Cultivation of rat pheochromocytoma cell line (PC12), as precursor cells of dopaminergic neural cells, on the scaffolds demonstrated that the introduction of MSNPs into PLGA and PLGA/gelatin matrix leads to improved cell attachment and proliferation and enhances cellular processes. - Highlights: • PLGA-based random nanofibers embedded with mesoporous silica nanoparticles were fabricated using electrospinning method • Incorporation of gelatin and MSNPs into PLGA-based scaffolds increased the hydrophilicity of scaffold • Addition of nanoparticles also improved the tensile mechanical properties of scaffolds • Introduction of MSNPs led to improved cell attachment and proliferation.

  10. A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells.

    Science.gov (United States)

    Hehnly, Heidi; Canton, David; Bucko, Paula; Langeberg, Lorene K; Ogier, Leah; Gelman, Irwin; Santana, L Fernando; Wordeman, Linda; Scott, John D

    2015-09-25

    Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin(-/-) mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division.

  11. Bone-Forming Capacity and Biodistribution of Bone Marrow-Derived Stromal Cells Directly Loaded Into Scaffolds: A Novel and Easy Approach for Clinical Application of Bone Regeneration.

    Science.gov (United States)

    Léotot, Julie; Lebouvier, Angélique; Hernigou, Philippe; Bierling, Philippe; Rouard, Hélène; Chevallier, Nathalie

    2015-01-01

    In the context of clinical applications of bone regeneration, cell seeding into scaffolds needs to be safe and easy. Moreover, cell density also plays a crucial role in the development of efficient bone tissue engineering constructs. The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure on hydroxyapatite/β-tricalcium phosphate (HA/βTCP), as well as define optimal cell density and control the biodistribution of grafted cells. To this end, human bone marrow-derived stromal cells (hBMSCs) were seeded on HA/βTCP scaffolds, and we have compared bone formation using an ectopic model. Our results demonstrated a significantly higher bone-forming capacity of hBMSCs directly loaded on HA/βTCP during surgery compared to hBMSCs preseeded for 7 days in vitro on HA/βTCP before ectopic implantation. The extent of new bone formation increases with increasing hBMSC densities quantitatively, qualitatively, and in frequency. Also, this study showed that grafted hBMSCs remained confined to the implantation site and did not spread toward other tissues, such as liver, spleen, lungs, heart, and kidneys. In conclusion, direct cell loading into a scaffold during surgery is more efficient for bone regeneration, as well as quick and safe. Therefore direct cell loading is suitable for clinical requirements and cell production control, making it a promising approach for orthopedic applications. Moreover, our results have provided evidence that the formation of a mature bone organ containing hematopoietic islets needs a sufficiently high local density of grafted hBMSCs, which should guide the optimal dose of cells for clinical use.

  12. Enhancing the Hydrophilicity and Cell Attachment of 3D Printed PCL/Graphene Scaffolds for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Weiguang Wang

    2016-12-01

    Full Text Available Scaffolds are physical substrates for cell attachment, proliferation, and differentiation, ultimately leading to the regeneration of tissues. They must be designed according to specific biomechanical requirements, i.e., certain standards in terms of mechanical properties, surface characteristics, porosity, degradability, and biocompatibility. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes, as well as surface treatment. Polymeric scaffolds reinforced with electro-active particles could play a key role in tissue engineering by modulating cell proliferation and differentiation. This paper investigates the use of an extrusion-based additive manufacturing system to produce poly(ε-caprolactone (PCL/pristine graphene scaffolds for bone tissue applications and the influence of chemical surface modification on their biological behaviour. Scaffolds with the same architecture but different concentrations of pristine graphene were evaluated from surface property and biological points of view. Results show that the addition of pristine graphene had a positive impact on cell viability and proliferation, and that surface modification leads to improved cell response.

  13. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells

    Directory of Open Access Journals (Sweden)

    Alain Carpentier

    2012-06-01

    Full Text Available Electrostimulation (ES can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 x 25 x 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002 and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01. Immunocytochemistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and bio- chemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  14. Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells.

    Science.gov (United States)

    Haneef, Kanwal; Lila, Nermine; Benadda, Samira; Legrand, Fabien; Carpentier, Alain; Chachques, Juan C

    2012-06-01

    Electrostimulation (ES) can be defined as a safe physical method to induce stem cell differentiation. The aim of this study is to evaluate the effectiveness of ES on bone marrow mesenchymal stem cells (BMSCs) seeded in collagen scaffolds in terms of proliferation and differentiation into cardiomyocytes. BMSCs were isolated from Wistar rats and seeded into 3D collagen type 1 templates measuring 25 × 25 × 6 mm. Bipolar in vitro ES was performed during 21 days. Electrical impedance and cell proliferation were measured. Expression of cardiac markers was assessed by immunocytochemistry. Viscoelasticity of collagen matrix was evaluated. Electrical impedance assessments showed a low resistance of 234±41 Ohms which indicates good electrical conductivity of collagen matrix. Cell proliferation at 570 nm as significantly increased in ES groups after seven day (ES 0.129±0.03 vs non-stimulated control matrix 0.06±0.01, P=0.002) and after 21 days, (ES 0.22±0.04 vs control 0.13±0.01, P=0.01). Immunocytoche mistry of BMSCs after 21 days ES showed positive staining of cardiac markers, troponin I, connexin 43, sarcomeric alpha-actinin, slow myosin, fast myosin and desmin. Staining for BMSCs marker CD29 after 21 days was negative. Electrostimulation of cell-seeded collagen matrix changed stem cell morphology and biochemical characteristics, increasing the expression of cardiac markers. Thus, MSC-derived differentiated cells by electrostimulation grafted in biological scaffolds might result in a convenient tissue engineering source for myocardial diseases.

  15. Electrospun Poly(l-lactide/Poly(ethylene glycol Scaffolds Seeded with Human Amniotic Mesenchymal Stem Cells for Urethral Epithelium Repair

    Directory of Open Access Journals (Sweden)

    Xiaokui Lv

    2016-08-01

    Full Text Available Tissue engineering-based urethral replacement holds potential for repairing large segmental urethral defects, which remains a great challenge at present. This study aims to explore the potential of combining biodegradable poly(l-lactide (PLLA/poly(ethylene glycol (PEG scaffolds and human amniotic mesenchymal cells (hAMSCs for repairing urethral defects. PLLA/PEG fibrous scaffolds with various PEG fractions were fabricated via electrospinning. The scaffolds were then seeded with hAMSCs prior to implantation in New Zealand male rabbits that had 2.0 cm-long defects in the urethras. The rabbits were randomly divided into three groups. In group A, hAMSCs were grown on PLLA/PEG scaffolds for two days and then implanted to the urethral defects. In group B, only the PLLA/PEG scaffolds were used to rebuild the rabbit urethral defect. In group C, the urethral defect was reconstructed using a regular urethral reparation technique. The repair efficacy was compared among the three groups by examining the urethral morphology, tissue reconstruction, luminal patency, and complication incidence (including calculus formation, urinary fistula, and urethral stricture using histological evaluation and urethral radiography methods. Findings from this study indicate that hAMSCs-loaded PLLA/PEG scaffolds resulted in the best urethral defect repair in rabbits, which predicts the promising application of a tissue engineering approach for urethral repair.

  16. Electrospun Poly(l-lactide)/Poly(ethylene glycol) Scaffolds Seeded with Human Amniotic Mesenchymal Stem Cells for Urethral Epithelium Repair

    Science.gov (United States)

    Lv, Xiaokui; Guo, Qianping; Han, Fengxuan; Chen, Chunyang; Ling, Christopher; Chen, Weiguo; Li, Bin

    2016-01-01

    Tissue engineering-based urethral replacement holds potential for repairing large segmental urethral defects, which remains a great challenge at present. This study aims to explore the potential of combining biodegradable poly(l-lactide) (PLLA)/poly(ethylene glycol) (PEG) scaffolds and human amniotic mesenchymal cells (hAMSCs) for repairing urethral defects. PLLA/PEG fibrous scaffolds with various PEG fractions were fabricated via electrospinning. The scaffolds were then seeded with hAMSCs prior to implantation in New Zealand male rabbits that had 2.0 cm-long defects in the urethras. The rabbits were randomly divided into three groups. In group A, hAMSCs were grown on PLLA/PEG scaffolds for two days and then implanted to the urethral defects. In group B, only the PLLA/PEG scaffolds were used to rebuild the rabbit urethral defect. In group C, the urethral defect was reconstructed using a regular urethral reparation technique. The repair efficacy was compared among the three groups by examining the urethral morphology, tissue reconstruction, luminal patency, and complication incidence (including calculus formation, urinary fistula, and urethral stricture) using histological evaluation and urethral radiography methods. Findings from this study indicate that hAMSCs-loaded PLLA/PEG scaffolds resulted in the best urethral defect repair in rabbits, which predicts the promising application of a tissue engineering approach for urethral repair. PMID:27517902

  17. Influence of Additive Manufactured Scaffold Architecture on the Distribution of Surface Strains and Fluid Flow Shear Stresses and Expected Osteochondral Cell Differentiation

    Science.gov (United States)

    Hendrikson, Wim J.; Deegan, Anthony J.; Yang, Ying; van Blitterswijk, Clemens A.; Verdonschot, Nico; Moroni, Lorenzo; Rouwkema, Jeroen

    2017-01-01

    Scaffolds for regenerative medicine applications should instruct cells with the appropriate signals, including biophysical stimuli such as stress and strain, to form the desired tissue. Apart from that, scaffolds, especially for load-bearing applications, should be capable of providing mechanical stability. Since both scaffold strength and stress–strain distributions throughout the scaffold depend on the scaffold’s internal architecture, it is important to understand how changes in architecture influence these parameters. In this study, four scaffold designs with different architectures were produced using additive manufacturing. The designs varied in fiber orientation, while fiber diameter, spacing, and layer height remained constant. Based on micro-CT (μCT) scans, finite element models (FEMs) were derived for finite element analysis (FEA) and computational fluid dynamics (CFD). FEA of scaffold compression was validated using μCT scan data of compressed scaffolds. Results of the FEA and CFD showed a significant impact of scaffold architecture on fluid shear stress and mechanical strain distribution. The average fluid shear stress ranged from 3.6 mPa for a 0/90 architecture to 6.8 mPa for a 0/90 offset architecture, and the surface shear strain from 0.0096 for a 0/90 offset architecture to 0.0214 for a 0/90 architecture. This subsequently resulted in variations of the predicted cell differentiation stimulus values on the scaffold surface. Fluid shear stress was mainly influenced by pore shape and size, while mechanical strain distribution depended mainly on the presence or absence of supportive columns in the scaffold architecture. Together, these results corroborate that scaffold architecture can be exploited to design scaffolds with regions that guide specific tissue development under compression and perfusion. In conjunction with optimization of stimulation regimes during bioreactor cultures, scaffold architecture optimization can be used to improve

  18. Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

    Science.gov (United States)

    Vikingsson, L; Claessens, B; Gómez-Tejedor, J A; Gallego Ferrer, G; Gómez Ribelles, J L

    2015-08-01

    In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior.

  19. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Science.gov (United States)

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-09-06

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application.

  20. Osteogenic induction of bone marrow-derived stromal cells on simvastatin-releasing, biodegradable, nano- to microscale fiber scaffolds.

    Science.gov (United States)

    Wadagaki, Ryu; Mizuno, Daiki; Yamawaki-Ogata, Aika; Satake, Makoto; Kaneko, Hiroaki; Hagiwara, Sumitaka; Yamamoto, Noriyuki; Narita, Yuji; Hibi, Hideharu; Ueda, Minoru

    2011-07-01

    Tissue engineering is an effective approach for the treatment of bone defects. Statins have been demonstrated to promote osteoblastic differentiation of bone marrow-derived stromal cells (BMSCs). Electrospun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano- to microscale topography and high porosity similar to the natural extracellular matrix (ECM). The aim of this study was to investigate the feasibility of a simvastatin-releasing, biodegradable, nano- to microscale fiber scaffold (SRBFS) for bone tissue engineering with BMSCs. Simvastatin was released from SRBFS slowly. BMSCs were observed to spread actively and rigidly adhere to SRBFS. BMSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture. Furthermore, osteoclastogenesis was suppressed by SRBFS in vitro. The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell-scaffold construct into an ectopic site on the murine back. These results suggest that SRBFS promoted osteoblastic differentiation of BMSCs in vitro and in vivo, and demonstrate feasibility as a bone engineering scaffold.

  1. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation

    Science.gov (United States)

    Zhou, Xuan; Castro, Nathan J.; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm2 intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  2. Preparation and investigation of polylactic acid, calcium carbonate and polyvinylalcohol nanofibrous scaffolds for osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    A. Doustgani

    2016-04-01

    Full Text Available Objective(s: In this study, the effect of electrospun fiber orientation on proliferation and differentiation of mesenchymal stem cells (MSCs was evaluated. Materials and Methods: Aligned and random nanocomposite nanofibrous scaffolds were electrospun from polylactic acid (PLA, poly (vinyl alcohol (PVA and calcium carbonate nanoparticles (nCaP. The surface morphology of prepared nanofibrous scaffolds with and without cell was examined using scanning electron microscopy. Mechanical properties of electrospun nanofibrous scaffolds were determined with a  universal testing machine. The in vitro properties of fabricated scaffolds was also investigated by the MTT assay and alkaline phosphatase activity (ALP.Results: The average fiber diameter for aligned and random nanofibers were 82 ± 12 nm and 124 ± 25 nm, respectively. The mechanical testing indicated the higher tensile strength and elastic modulus of aligned nanofibers. MTT and ALP results showed that alignment of nanofiber increased the osteogenic differentiation of stem cells.Conclusion: Aligned nanofibrous nanocomposite scaffolds of PLA/nCaP/PVA could be an excellent substrate for MSCs and represents a potential bone-filling material.

  3. Multifunctional chitosan/polyvinyl pyrrolidone/45S5 Bioglass® scaffolds for MC3T3-E1 cell stimulation and drug release

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Qingqing [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Li, Wei [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Yu, Shanshan; Ma, Liwei [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Jin, Dayong [Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, NSW 2007 (Australia); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia); Boccaccini, Aldo R., E-mail: Aldo.Boccaccini@ww.uni-erlangen.de [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstrasse 6, Erlangen 91058 (Germany); Liu, Yong, E-mail: yongliu1980@hotmail.com [Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Wenzhou Medical University, 270 Xueyuan Xi Road, Wenzhou, Zhejiang 325027 (China); Advanced Cytometry Labs, ARC Center of Excellence for Nanoscale BioPhotonics, Macquarie University, Sydney, NSW 2109 (Australia)

    2015-11-01

    Novel chitosan–polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24 h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7 days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering. - Highlights: • Novel genipi–chitosan–polyvinyl pyrrolidone/45S5 Bioglass® scaffolds are prepared. • Resistance to enzymatic degradation of the scaffold is improved significantly. • The resulting scaffold shows enhanced MC3T3-E1 cell adhesion and proliferation. • Release of antibiotic vancomycin from the

  4. Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

    Science.gov (United States)

    Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

    2017-01-16

    Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

  5. Evaluation of Three-Dimensional Porous Iron-Cross-Linked Alginate as a Scaffold for Cell Culture

    OpenAIRE

    Machida-Sano, Ikuko; Ogawa, Sakito; Hirakawa, Makoto; Namiki, Hideo

    2014-01-01

    We investigated the efficacy of three-dimensional porous ferric-ion-cross-linked alginate (Fe-alginate) gels as cell scaffolds, in comparison with calcium-ion-cross-linked alginate (Ca-alginate) gels. In a previous study, we had demonstrated that two-dimensional Fe-alginate film was an efficient material for use as a scaffold, allowing good cell adhesion and proliferation, unlike Ca-alginate film. In the present study, we fabricated three-dimensional porous Fe- and Ca-alginate gels by freeze-...

  6. Bioactive glass-based composites for the production of dense sintered bodies and porous scaffolds.

    Science.gov (United States)

    Bellucci, D; Sola, A; Cannillo, V

    2013-05-01

    Recently several attempts have been made to combine calcium phosphates, such as β-tricalcium phosphate (β-TCP) and, most of all, hydroxyapatite (HA), with bioactive glasses of different composition, in order to develop composites with improved biological and mechanical performance. Unfortunately, the production of such systems usually implies a high-temperature treatment (up to 1300 °C), which may result in several drawbacks, including crystallization of the original glass, decomposition of the calcium phosphate phase and/or reactions between the constituent phases, with non-trivial consequences in terms of microstructure, bioactivity and mechanical properties of the final samples. In the present contribution, novel binary composites have been obtained by sintering a bioactive glass, characterized by a low tendency to crystallize, with the addition of HA or β-TCP as the second phase. In particular, the composites have been treated at a relatively low temperature (818 °C and 830 °C, depending on the sample), thus preserving the amorphous structure of the glass and minimizing the interaction between the constituent phases. The effects of the glass composition, calcium phosphate nature and processing conditions on the composite microstructure, mechanical properties and in vitro bioactivity have been systematically discussed. To conclude, a feasibility study to obtain scaffolds for bone tissue regeneration has been proposed.

  7. Novel template-casting technique for fabricating beta-tricalcium phosphate scaffolds with high interconnectivity and mechanical strength and in vitro cell responses.

    Science.gov (United States)

    Liu, Yongxing; Kim, Joong-Hyun; Young, Daniel; Kim, Sungwoo; Nishimoto, Satoru K; Yang, Yunzhi

    2010-03-01

    A novel template-casting method was developed to produce completely interconnected, macroporous biodegradable beta-tricalcium phosphate (beta-TCP) scaffolds, whose architecture and chemistry can be fully manipulated by varying the templates and casting materials. The processing route includes preparation of beta-TCP slurry; casting and shaping into preformed templates comprised of paraffin beads; solidifying, drying; and sintering. Structural, chemical, and mechanical properties of the prepared macroporous scaffolds were characterized using micro computed tomography, scanning electron microscopy, x-ray diffractometry, Fourier transform infrared spectroscopy, and mechanical testing. Human embryonic palatal mesenchymal cells were used to evaluate cell proliferation within the scaffolds in vitro. The scaffolds consisted of interconnected macropores and solid struts, leading to a reticular network. Two groups of scaffolds with larger pores, approximately 600-800 microm and smaller pores approximately 350-500 microm, were demonstrated. The interconnected windows between neighboring macropores were 440 +/- 57 microm in diameter for the larger-pored scaffolds, and 330 +/- 50 microm for the smaller-pored scaffolds. The scaffolds were highly crystallized and composed dominantly of beta-tricalcium phosphate (beta-TCP) accompanied by minor phase of hydroxyapatite (HA). The hydroxyl group was clearly detected by FTIR on the scaffolds. High mechanical strength (9.3 MPa) was demonstrated by the completely interconnected scaffolds with approximately 79% porosity. The human embryonic palatal mesenchymal (HEPM) cells proliferated well on the smaller-pored and larger-pored scaffolds, exhibiting a significantly higher level of proliferation in the first 11 days of culture on the smaller pored scaffolds. High levels of differentiation were also evidenced in both pore sizes of scaffolds.

  8. The influence of electrospun fibre scaffold orientation and nano-hydroxyapatite content on the development of tooth bud stem cells in vitro

    NARCIS (Netherlands)

    Manen, E.H. van; Zhang, W.; Walboomers, X.F.; Vazquez, B.; Yang, F.; Ji, W.; Yu, N.; Spear, D.J.; Jansen, J.A.; Yelick, P.C.

    2014-01-01

    In stem cell-based dental tissue engineering, the goal is to create tooth-like structures using scaffold materials to guide the dental stem cells. In this study, the effect of fiber alignment and hydroxyapatite content in biodegradable electrospun PLGA scaffolds have been investigated. Fiber orienta

  9. Evaluation of early healing events around mesenchymal stem cell-seeded collagen-glycosaminoglycan scaffold. An experimental study in Wistar rats.

    LENUS (Irish Health Repository)

    Alhag, Mohamed

    2011-03-01

    Tissue engineering using cell-seeded biodegradable scaffolds offers a new bone regenerative approach that might circumvent many of the limitations of current therapeutic modalities. The aim of this experiment was to study the early healing events around mesenchymal stem cell-seeded collagen-glycosaminoglycan scaffolds.

  10. Repetitive Arg-Gly-Asp peptide as a cell-stimulating agent on electrospun poly(ϵ-caprolactone) scaffold for tissue engineering.

    Science.gov (United States)

    Chaisri, Pacharaporn; Chingsungnoen, Artit; Siri, Sineenat

    2013-11-01

    Electrospun scaffolds derived from poly(ϵ-caprolactone) (PCL), a well known biodegradable material, have an architecture that is suitable for hosting cells. However, their biomedical applications are restricted because these scaffolds lack the bioactivity necessary to stimulate cell responses. In this work, a repetitive Arg-Gly-Asp (rRGD) peptide was produced as a cell-stimulating agent to provide the PCL scaffold with bioactivity. DNA encoding rRGD was amplified by polymerase chain reaction using overlap primers without a DNA template, and cloned into a protein expression vector to produce a His-tag fusion peptide. In an in vitro cell adhesion assay, the purified rRGD peptide, comprising 30 RGD repeats, promoted a 1.5-fold greater cell adhesion than the commercial tripeptide RGD. The rRGD peptide was immobilized onto an electrospun PCL scaffold that had been pretreated with argon plasma and graft-polymerized with acrylic acid. Fourier transform infrared (FTIR) analysis indicated that covalently linked rRGD peptide was present on the scaffold. The PCL scaffold with immobilized rRGD showed significantly changed hydrophilic properties and an enhanced adhesion and proliferation of mouse fibroblast cells by 2.3- and 2.9-fold, respectively, compared to the PCL scaffold alone. Through its ability to promote cell adhesion and proliferation, the rRGD peptide has great potential as a stimulant for improving the suboptimal cell-matrix interaction of polymeric scaffolds for tissue engineering applications.

  11. Fatigue and human umbilical cord stem cell seeding characteristics of calcium phosphate-chitosan-biodegradable fiber scaffolds.

    Science.gov (United States)

    Zhao, Liang; Burguera, Elena F; Xu, Hockin H K; Amin, Nikhil; Ryou, Heon; Arola, Dwayne D

    2010-02-01

    Calcium phosphate cement (CPC) has in situ-setting ability and bioactivity, but the brittleness and low strength limit CPC to only non-load-bearing bone repairs. Human umbilical cord mesenchymal stem cells (hUCMSCs) can be harvested without an invasive procedure required for the commonly studied bone marrow MSCs. However, little has been reported on hUCMSC delivery via bioactive scaffolds for bone tissue engineering. The objectives of this study were to develop CPC scaffolds with improved resistance to fatigue and fracture, and to investigate hUCMSC delivery for bone tissue engineering. In fast fracture, CPC with 15% chitosan and 20% polyglactin fibers (CPC-chitosan-fiber scaffold) had flexural strength of 26mPa, higher than 10mPa for CPC control (pfiber specimens that survived 2x10(6) cycles had the maximum stress of 10MPa, compared to 5MPa of CPC control. CPC-chitosan-fiber specimens that failed after multiple cycles had a mean stress-to-failure of 9MPa, compared to 5.8MPa for CPC control (pfiber scaffolds. The percentage of live cells reached 96-99%. Cell density was about 300cells/mm(2) at day 1; it proliferated to 700cells/mm(2) at day 4. Wst-1 assay showed that the stronger CPC-chitosan-fiber scaffold had hUCMSC viability that matched the CPC control (p>0.1). In summary, this study showed that chitosan and polyglactin fibers substantially increased the fatigue resistance of CPC, and that hUCMSCs had excellent proliferation and viability on the scaffolds.

  12. Novel thin films deposited on electrospun PCL scaffolds by atmospheric pressure plasma jet for L929 fibroblast cell cultivation

    Science.gov (United States)

    Gozutok, M.; Baitukha, A.; Arefi-Khonsari, F.; Turkoglu Sasmazel, H.

    2016-11-01

    This paper reports on the deposition of PCL homopolymers and poly ɛ-caprolactone-polyethylene glycol (PCL-PEG) copolymers by atmospheric pressure plasma jet (APPJ) onto electrospun PCL scaffolds for improving L929 fibroblast cell growth. Polymer deposited scaffolds showed better stability as well as lower CA as compared to those treated with APPJ in Ar alone used as the carrier gas to introduce the precursors due to the formation of polar groups generated during the plasma treatment, such as -OH and/or -COO. Average fiber and porosity sizes were calculated by using SEM photographs and the ImageJ Launcher Software program and higher values were observed for both PCL and PCL-PEG deposited scaffolds than the untreated electrospun PCL scaffolds. XPS analysis showed that C1s% content decreased for PCL deposited (from 82.4% to 71.0%) and PCL-PEG deposited (from 82.4% to 57.7%) and O1s% composition increased for PCL deposited (from 17.6% to 29.0%) and PCL-PEG deposited (from 17.6% to 42.3%) compared to the untreated one. XPS results proved more incorporation of oxygen moieties on the deposited surfaces than the untreated samples giving rise to more hydrophilic surfaces to the deposited ones. Standard in vitro MTT test, Giemsa staining, fluorescence and CLSM imaging techniques were used for the determination of cell viability, adhesion and proliferation. Cell culture experiments showed that PCL-PEG deposited electrospun PCL scaffolds had the most promising cell adhesion, proliferation and growth among the treated scaffolds. The increased average fiber diameter caused by deposition as well as oxygen containing polar groups formed on the surfaces due to the radicals present in the plasma atmosphere provided higher surface area and functionality, respectively, for cells to attach, yielding better biocompatibility performance.

  13. Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

    Science.gov (United States)

    Guillaume, Olivier; Naqvi, Syeda Masooma; Lennon, Kerri; Buckley, Conor Timothy

    2015-04-01

    Lower lumbar disc disorders pose a significant problem in an aging society with substantial socioeconomic consequences. Both inner tissue (nucleus pulposus) and outer tissue (annulus fibrosus) of the intervertebral disc are affected by such debilitating disorders and can lead to disc herniation and lower back pain. In this study, we developed an alginate-collagen composite porous scaffold with shape-memory properties to fill defects occurring in annulus fibrosus tissue of degenerated intervertebral discs, which has the potential to be administered using minimal invasive surgery. In the first part of this work, we assessed how collagen incorporation on preformed alginate scaffolds influences the physical properties of the final composite scaffold. We also evaluated the ability of annulus fibrosus cells to attach, migrate, and proliferate on the composite alginate-collagen scaffolds compared to control scaffolds (alginate only). In vitro experiments, performed in intervertebral disc-like microenvironmental conditions (low glucose and low oxygen concentrations), revealed that for alginate only scaffolds, annulus fibrosus cells agglomerated in clusters with limited infiltration and migration capacity. In comparison, for alginate-collagen scaffolds, annulus fibrosus cells readily attached and colonized constructs, while preserving their typical fibroblastic-like cell morphology with spreading behavior and intense cytoskeleton expression. In a second part of this study, we investigated the effects of alginate-collagen scaffold when seeded with bone marrow derived mesenchymal stem cells. In vitro, we observed that alginate-collagen porous scaffolds supported cell proliferation and extracellular matrix deposition (collagen type I), with secretion amplified by the local release of transforming growth factor-β3. In addition, when cultured in ex vivo organ defect model, alginate-collagen scaffolds maintained viability of transplanted mesenchymal stem cells for up to 5

  14. Patterned carbon nanotubes as a new three-dimensional scaffold for mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Bitirim, Verda Ceylan [Department of Molecular Biology and Genetics, Bilkent University, 06800, Ankara (Turkey); Kucukayan-Dogu, Gokce [Institute of Engineering and Science, Material Science and Nanotechnology Graduate Program, Bilkent University, 06800, Ankara (Turkey); Bengu, Erman [Department of Chemistry, Bilkent University, 06800, Ankara (Turkey); Akcali, Kamil Can, E-mail: akcali@fen.bilkent.edu.tr [Department of Molecular Biology and Genetics, Bilkent University, 06800, Ankara (Turkey); Bilgen, Bilkent University Genetics and Biotechnology Research Center, 06800, Ankara (Turkey)

    2013-07-01

    We investigated the cellular adhesive features of mesenchymal stem cells (MSC) on non-coated and collagen coated patterned and vertically aligned carbon nanotube (CNT) structures mimicking the natural extra cellular matrix (ECM). Patterning was achieved using the elasto-capillary induced by water treatment on the CNT arrays. After confirmation with specific markers both at transcript and protein levels, MSCs from different passages were seeded on either collagen coated or non-coated patterned CNTs. Adhesion and growth of MSCs on the patterned CNT arrays were examined using scanning electron microscopy image analysis and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. The highest MSC count was observed on the non-coated patterned CNTs at passage zero, while decreasing numbers of MSCs were found at the later passages. Similarly, MTT assay results also revealed a decrease in the viability of the MSCs for the later passages. Overall, the cell count and viability experiments indicated that MSCs were able to better attach to non-coated patterned CNTs compared to those coated with collagen. Therefore, the patterned CNT surfaces can be potentially used as a scaffold mimicking the ECM environment for MSC growth which presents an alternative approach to MSC-based transplantation therapy applications. - Highlights: • Synthesized vertically aligned CNTs were patterned to be used as scaffold. • The growth of mesenchymal stem cells was achieved on the patterned CNTs. • The cell number was counted higher on the patterned CNTs than collagen coated CNTs. • The MTT assay results revealed the cell viability on the patterned CNTs.

  15. The Biological Function of the Prion Protein: A Cell Surface Scaffold of Signaling Modules

    Science.gov (United States)

    Linden, Rafael

    2017-01-01

    The prion glycoprotein (PrPC) is mostly located at the cell surface, tethered to the plasma membrane through a glycosyl-phosphatydil inositol (GPI) anchor. Misfolding of PrPC is associated with the transmissible spongiform encephalopathies (TSEs), whereas its normal conformer serves as a receptor for oligomers of the β-amyloid peptide, which play a major role in the pathogenesis of Alzheimer’s Disease (AD). PrPC is highly expressed in both the nervous and immune systems, as well as in other organs, but its functions are controversial. Extensive experimental work disclosed multiple physiological roles of PrPC at the molecular, cellular and systemic levels, affecting the homeostasis of copper, neuroprotection, stem cell renewal and memory mechanisms, among others. Often each such process has been heralded as the bona fide function of PrPC, despite restricted attention paid to a selected phenotypic trait, associated with either modulation of gene expression or to the engagement of PrPC with a single ligand. In contrast, the GPI-anchored prion protein was shown to bind several extracellular and transmembrane ligands, which are required to endow that protein with the ability to play various roles in transmembrane signal transduction. In addition, differing sets of those ligands are available in cell type- and context-dependent scenarios. To account for such properties, we proposed that PrPC serves as a dynamic platform for the assembly of signaling modules at the cell surface, with widespread consequences for both physiology and behavior. The current review advances the hypothesis that the biological function of the prion protein is that of a cell surface scaffold protein, based on the striking similarities of its functional properties with those of scaffold proteins involved in the organization of intracellular signal transduction pathways. Those properties are: the ability to recruit spatially restricted sets of binding molecules involved in specific signaling

  16. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials.

    Science.gov (United States)

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang

    2011-07-21

    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials.

  17. The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs.

    LENUS (Irish Health Repository)

    Lyons, Frank G

    2010-12-01

    One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.

  18. Growth factor and small molecule influence on urological tissue regeneration utilizing cell seeded scaffolds.

    Science.gov (United States)

    Sharma, Arun K; Cheng, Earl Y

    2015-03-01

    Regenerative medicine strategies combine various attributes from multiple disciplines including stem cell biology, chemistry, materials science and medicine. The junction at which these disciplines intersect provides a means to address unmet medical needs in an assortment of pathologies with the goal of creating sustainable, functional replacement tissues. Tissue damage caused by trauma for example, requires rapid responses in order to mitigate further tissue deterioration. Cell/scaffold composites have been utilized to initiate and stabilize regenerative responses in vivo with the hope that functional tissue can be attained. Along with the gross reconfiguration of regenerating tissues, small molecules and growth factors also play a pivotal role in tissue regeneration. Several regenerative studies targeting a variety of urological tissues demonstrate the utility of these small molecules or growth factors in an in vivo setting.

  19. Poly(amidoamine Hydrogels as Scaffolds for Cell Culturing and Conduits for Peripheral Nerve Regeneration

    Directory of Open Access Journals (Sweden)

    Fabio Fenili

    2011-01-01

    Full Text Available Biodegradable and biocompatible poly(amidoamine-(PAA- based hydrogels have been considered for different tissue engineering applications. First-generation AGMA1 hydrogels, amphoteric but prevailing cationic hydrogels containing carboxylic and guanidine groups as side substituents, show satisfactory results in terms of adhesion and proliferation properties towards different cell lines. Unfortunately, these hydrogels are very swellable materials, breakable on handling, and have been found inadequate for other applications. To overcome this problem, second-generation AGMA1 hydrogels have been prepared adopting a new synthetic method. These new hydrogels exhibit good biological properties in vitro with satisfactory mechanical characteristics. They are obtained in different forms and shapes and successfully tested in vivo for the regeneration of peripheral nerves. This paper reports on our recent efforts in the use of first-and second-generation PAA hydrogels as substrates for cell culturing and tubular scaffold for peripheral nerve regeneration.

  20. FGL-functionalized self-assembling nanofiber hydrogel as a scaffold for spinal cord-derived neural stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jian [Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng, Jin [Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng, Qixin, E-mail: zheng-qx@163.com [Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Wu, Yongchao; Wu, Bin; Huang, Shuai; Fang, Weizhi; Guo, Xiaodong [Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China)

    2015-01-01

    A class of designed self-assembling peptide nanofiber scaffolds has been shown to be a good biomimetic material in tissue engineering. Here, we specifically made a new peptide hydrogel scaffold FGLmx by mixing the pure RADA{sub 16} and designer functional peptide RADA{sub 16}-FGL solution, and we analyzed the physiochemical properties of each peptide with atomic force microscopy (AFM) and circular dichroism (CD). In addition, we examined the biocompatibility and bioactivity of FGLmx as well as RADA{sub 16} scaffold on spinal cord-derived neural stem cells (SC-NSCs) isolated from neonatal rats. Our results showed that RADA{sub 16}-FGL displayed a weaker β-sheet structure and FGLmx could self-assemble into nanofibrous morphology. Moreover, we found that FGLmx was not only noncytotoxic to SC-NSCs but also promoted SC-NSC proliferation and migration into the three-dimensional (3-D) scaffold, meanwhile, the adhesion and lineage differentiation of SC-NSCs on FGLmx were similar to that on RADA{sub 16}. Our results indicated that the FGL-functionalized peptide scaffold might be very beneficial for tissue engineering and suggested its further application for spinal cord injury (SCI) repair. - Highlights: • RADA{sub 16} and RADA{sub 16}-FGL peptides were synthesized and characterized. • Rat spinal cord neural stem cells were successfully isolated and characterized. • We provided an induction method for mixed differentiation of neural stem cells. • FGL scaffold had good biocompatibility and bioactivity with neural stem cells.

  1. Mild hypothermia combined with a scaffold of NgR-silenced neural stem cells/Schwann cells to treat spinal cord injur y

    Institute of Scientific and Technical Information of China (English)

    Dong Wang; Jinhua Liang; Jianjun Zhang; Shuhong Liu; Wenwen Sun

    2014-01-01

    Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differenti-ation, a cell-scaffold complex seeded with Nogo receptor (NgR)-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previ-ous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly(D,L-lactide-co-glycolic acid) (PLGA) scaffold seeded with NgR-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the NgR-silencing cells+PLGA group, hindlimb motor function and nerve electrophysiological function were clearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve ifbers were increased in the group treated with the NgR-silenced cell scaffold+mild hypothermia at 34°C for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviv-ing cells and nerve ifbers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with NgR gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury.

  2. Wnt11 plays an important role in the osteogenesis of human mesenchymal stem cells in a PHA/FN/ALG composite scaffold: possible treatment for infected bone defect

    OpenAIRE

    Wang, Hai; He, Xiao-Qing; Jin, Tao; Li, Yang; Fan, Xin-Yu; Wang, Yi; Xu, Yong-Qing

    2016-01-01

    Background Infected bone defect poses a great challenge for orthopedists because it is difficult to cure. Tissue-engineered bone based on the human mesenchymal stem cells (hMSCs), has currently taken a promising treatment protocol in clinical practice. In a previous study, a porous hydroxyapatite/fibronectin/alginate (PHA/FN/ALG) composite scaffold displayed favorable biological properties as a novel scaffold, which was considered better than single-material scaffolds. In addition, Wnt11 has ...

  3. Differentiation of Dental Pulp Stem Cells on Gutta-Percha Scaffolds

    Directory of Open Access Journals (Sweden)

    Liudi Zhang

    2016-05-01

    Full Text Available Advances in treatment of tooth injury have shown that tooth regeneration from the pulp was a viable alternative of root canal therapy. In this study, we demonstrated that Gutta-percha, nanocomposites primarily used for obturation of the canal, are not cytotoxic and can induce differentiation of dental pulp stem cells (DPSC in the absence of soluble mediators. Flat scaffolds were obtained by spin coating Si wafers with three Gutta-percha compounds: GuttaCore™, ProTaper™, and Lexicon™. The images of annealed surfaces showed that the nanoparticles were encapsulated, forming surfaces with root mean square (RMS roughness of 136–211 nm. Then, by culturing DPSC on these substrates we found that after some initial difficulty in adhesion, confluent tissues were formed after 21 days. Imaging of the polyisoprene (PI surfaces showed that biomineral deposition only occurred when dexamethasone was present in the media. Spectra obtained from the minerals was consistent with that of hydroxyapatite (HA. In contrast, HA deposition was observed on all Gutta-percha scaffolds regardless of the presence or absence of dexamethasone, implying that surface roughness may be an enabling factor in the differentiation process. These results indicate that Gutta-percha nanocomposites may be good candidates for pulp regeneration therapy.

  4. Three-dimensional imaging of the developing vasculature within stem cell-seeded scaffolds cultured in ovo

    Directory of Open Access Journals (Sweden)

    Anna eWoloszyk

    2016-04-01

    Full Text Available Successful tissue engineering requires functional vascularization of the three-dimensional constructs with the aim to serve as implants for tissue replacement and regeneration. The survival of the implant is only possible if the supply of oxygen and nutrients by developing capillaries from the host is established. The chorioallantoic membrane assay is a valuable tool to study the ingrowth and distribution of vessels into scaffolds composed by appropriate biomaterials and stem cell populations that are used in cell-based regenerative approaches. The developing vasculature of chicken embryos within cell-seeded scaffolds can be visualized with microcomputed tomography after intravenous injection of MicroFil®, which is a radiopaque contrast agent. Here, we provide a step-by-step protocol for the seeding of stem cells into silk fibroin scaffolds, the chorioallantoic membrane culture conditions, the procedure of MicroFil® perfusion, and finally the microcomputed tomography scanning. Three-dimensional imaging of the vascularized tissue engineered constructs provides an important analytical tool for studying the potential of cell seeded scaffolds to attract vessels and form vascular networks, as well as for analyzing the number, density, length, branching, and diameter of vessels. This in ovo method can greatly help to screen implants that will be used for tissue regeneration purposes before their in vivo testing, thereby reducing the amount of animals needed for pre-clinical studies.

  5. Hyaluronan and Fibrin Biomaterial as Scaffolds for Neuronal Differentiation of Adult Stem Cells Derived from Adipose Tissue and Skin

    Directory of Open Access Journals (Sweden)

    Chiara Gardin

    2011-10-01

    Full Text Available Recently, we have described a simple protocol to obtain an enriched culture of adult stem cells organized in neurospheres from two post-natal tissues: skin and adipose tissue. Due to their possible application in neuronal tissue regeneration, here we tested two kinds of scaffold well known in tissue engineering application: hyaluronan based membranes and fibrin-glue meshes. Neurospheres from skin and adipose tissue were seeded onto two scaffold types: hyaluronan based membrane and fibrin-glue meshes. Neurospheres were then induced to acquire a glial and neuronal-like phenotype. Gene expression, morphological feature and chromosomal imbalance (kariotype were analyzed and compared. Adipose and skin derived neurospheres are able to grow well and to differentiate into glial/neuron cells without any chromosomal imbalance in both scaffolds. Adult cells are able to express typical cell surface markers such as S100; GFAP; nestin; βIII tubulin; CNPase. In summary, we have demonstrated that neurospheres isolated from skin and adipose tissues are able to differentiate in glial/neuron-like cells, without any chromosomal imbalance in two scaffold types, useful for tissue engineering application: hyaluronan based membrane and fibrin-glue meshes.

  6. In Vivo Differentiation of Mesenchymal Stem Cells into Insulin Producing Cells on Electrospun Poly-L-Lactide Acid Scaffolds Coated with Matricaria chamomilla L. Oil

    Directory of Open Access Journals (Sweden)

    Afsaneh Fazili

    2016-09-01

    Full Text Available Objective: This study examined the in vivo differentiation of mesenchymal stem cells (MSCs into insulin producing cells (IPCs on electrospun poly-L-lactide acid (PLLA scaffolds coated with Matricaria chammomila L. (chamomile oil. Materials and Methods: In this interventional, experimental study adipose MSCs (AMSCs were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control or PLLA scaffold coated with chamomile oil (experimental. A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs. Real-time polymerase chain reaction (PCR determined the expressions of genes related to pancreatic endocrine development and function. Results: Fourier transform infrared spectroscopy (FTIR results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm-1 for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001 compared to control group (0.063 ± 0.009, P≤0.05. Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation. Conclusion: The pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular

  7. Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

    Institute of Scientific and Technical Information of China (English)

    Yuping Feng; Jiao Wang; Shixin Ling; Zhuo Li; Mingsheng Li; Qiongyi Li; Zongren Ma; Sijiu Yu

    2014-01-01

    The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

  8. Odontogenic cell culture in PEGDA hydrogel scaffolds for use in tooth regeneration protocols.

    Science.gov (United States)

    Jaramillo, Lorenza; Briceño, Ignacio; Durán, Camilo

    2012-01-01

    In order to obtain a tooth-like structure, embryonic oral ectoderm cells (EOE) and bone marrow-derived stem cells (BMSC) were stratified within a synthetic hydrogel matrix (PEGDA) and implanted in the ileal mesentery of adult male Lewis rats. Whole-mount in situ hybridization was used to evaluate the expression of Pitx2, Shh and Wnt10a signals indicative of tooth initiation. In rats, expression of the three markers was present in the oral ectoderm starting at embryonic stage E12.5. which was therefore selected for cell harvesting. Embryos were obtained by controlled service of young female Lewis rats in which estrus was detected by impedance reading. At E12.5, pregnant rats were humanely euthanized and embryos were collected. The mandibular segment of the first branchial arch was dissected and the mesenchyme separated from the ectoderm by enzymatic digestion with pancreatin trypsin solution. BMSCs were collected by flushing the marrow of tibiae and femurs of adult Lewis rats with alpha-MEM and cultured in alpha-MEM in 25 cm2 flasks. Second passage BMSC's were recombined with competent oral ectoderm (E12.5-E13) stratifying them within a 3D PEGDA scaffold polymerized by exposure to UV (365 nm) inside a pyramidal polypropylene mold. Constructs were incubated from 24 to 48 hrs in alpha-MEM and then implanted for four to six weeks in the mesentery of adult male (3-6 month old) Lewis rats. 76 constructs were implanted (37 experimental, 27 negative controls and 12 positive controls). Upon maturation, constructs were harvested, fixed in buffered formalin, processed and stained with hematoxylin eosin (HE). Histological evaluation of the experimental and negative constructs showed that BMSCs underwent an apoptotic process due to lack of matrix interactions, known as anoikis, and were thus incapable of interacting with the competent ectoderm. In contrast, embryonic oral ectoderm was able to proliferate during the mesenteric implantation. In conclusion, PEGDA scaffolds are

  9. Gel de plaquetas: arcabouço 3D para cultura celular Platelet gel: 3D scaffold for cell culture

    Directory of Open Access Journals (Sweden)

    Andrei Moroz

    2009-01-01

    conditions, observed at an inverted microscope on a daily basis. RESULTS: Both forms were successful because they produced a 3D environment that supports cell growth, with positive and negative features. DISCUSSION: The Sphere form didn't attach to the plate. Gel retraction was observed and the investigation at the microscope was difficult, because of the opaque areas in the optical field. The Carpet form didn't retract, and didn't produce opaque areas. Follow-up time was 20 days. CONCLUSIONS: The production of a PRP 3D scaffold was successful, and this is an alternative requiring further investigation in order to establish an efficient and reliable route in tissue engineering technology, particularly in cartilage tissue culture.

  10. Bio-safe processing of polylactic-co-caprolactone and polylactic acid blends to fabricate fibrous porous scaffolds for in vitro mesenchymal stem cells adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Salerno, Aurelio, E-mail: asalerno@unina.it [Centre for Advanced Biomaterials for Health Care, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, 80125 Napoli (Italy); Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus de la UAB s/n, Bellaterra 08193 (Spain); Guarino, Vincenzo; Oliviero, Olimpia; Ambrosio, Luigi [Institute of Polymers, Composites and Biomaterials, National Research Council of Italy, V.le Kennedy 54, Pad 20, Mostra d' Oltremare, 80125 Naples (Italy); Domingo, Concepción [Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus de la UAB s/n, Bellaterra 08193 (Spain)

    2016-06-01

    In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-caprolactone (PLC) and polylactic acid (PLA) polymers, for tissue engineering applications is reported. The scaffolds are prepared by means of a bio-safe thermally induced phase separation (TIPS) approach with or without the addition of NaCl particles used as particulate porogen. The scaffolds are characterized to assess their crystalline structure, morphology and mechanical properties, and the texture of the pores and the pore size distribution. Moreover, in vitro human mesenchymal stem cells (hMSCs) culture tests have been carried out to demonstrate the biocompatibility of the scaffolds. The results of this study demonstrate that all of the scaffold materials processed by means of TIPS process are semi-crystalline. Furthermore, the blend composition affected polymer crystallization and, in turn, the nano and macro-structural properties of the scaffolds. Indeed, neat PLC and neat PLA crystallize into globular and randomly arranged sub micro-size scale fibrous conformations, respectively. Concomitantly, the addition of NaCl particles during the fabrication route allows for the creation of an interconnected network of large pores inside the primary structure while resulted in a significant decrease of scaffolds mechanical response. Finally, the results of cell culture tests demonstrate that both the micro and macro-structure of the scaffold affect the in vitro hMSCs adhesion and proliferation. - Highlights: • Porous scaffolds are prepared by polymer blending, phase separation and NaCl leaching. • The process avoids the use of toxic solvents. • Blend composition dictates polymer crystallization and scaffold properties. • Scaffolds are provided of a sub micro-scale fibers structure and interconnected macropores. • Stem cells adhesion and proliferation depend on scaffolds composition and structure.

  11. In vitro comparative study of white and dark polycaprolactone trifumarate in situ cross-linkable scaffolds seeded with rat bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Kama Bistari Muhammad

    2012-01-01

    Full Text Available OBJECTIVE: Dark poly(caprolactone trifumarate is a successful candidate for use as a bone tissue engineering scaffold. Recently, a white polymeric scaffold was developed that shows a shorter synthesis time and is more convenient for tissue-staining work. This is an in vitro comparative study of both the white and dark scaffolds. METHODS: Both white and dark poly(caprolactone trifumarate macromers were characterized via Fourier transform infrared spectroscopy before being chemically cross-linked and molded into disc-shaped scaffolds. Biodegradability was assessed by percentage weight loss on days 7, 14, 28, 42 and 56 (n = 5 after immersion in 10% serum-supplemented medium or distilled water. Static cell seeding was employed in which isolated and characterized rat bone marrow stromal cells were seeded directly onto the scaffold surface. Seeded scaffolds were subjected to a series of biochemical assays and scanning electron microscopy at specified time intervals for up to 28 days of incubation. RESULTS: The degradation of the white scaffold was significantly lower compared with the dark scaffold but was within the acceptable time range for bone-healing processes. The deoxyribonucleic acid and collagen contents increased up to day 28 with no significant difference between the two scaffolds, but the glycosaminoglycan content was slightly higher in the white scaffold throughout 14 days of incubation. Scanning electron microscopy at days 1 and 14 revealed cellular growth and attachment. CONCLUSIONS: There was no cell growth advantage between the two forms, but the white scaffold had a slower biodegradability rate, suggesting that the newly synthesized poly(caprolactone trifumarate is more suitable for use as a bone tissue engineering scaffold.

  12. Scaffold-based delivery of autologous mesenchymal stem cells for mandibular distraction osteogenesis: preliminary studies in a porcine model.

    Directory of Open Access Journals (Sweden)

    Zongyang Sun

    Full Text Available PURPOSE: Bone regeneration through distraction osteogenesis (DO is promising but remarkably slow. To accelerate it, autologous mesenchymal stem cells have been directly injected to the distraction site in a few recent studies. Compared to direct injection, a scaffold-based method can provide earlier cell delivery with potentially better controlled cell distribution and retention. This pilot project investigated a scaffold-based cell-delivery approach in a porcine mandibular DO model. MATERIALS AND METHODS: Eleven adolescent domestic pigs were used for two major sets of studies. The in-vitro set established methodologies to: aspirate bone marrow from the tibia; isolate, characterize and expand bone marrow-derived mesenchymal stem cells (BM-MSCs; enhance BM-MSC osteogenic differentiation using FGF-2; and confirm cell integration with a gelatin-based Gelfoam scaffold. The in-vivo set transplanted autologous stem cells into the mandibular distraction sites using Gelfoam scaffolds; completed a standard DO-course and assessed bone regeneration by macroscopic, radiographic and histological methods. Repeated-measure ANOVAs and t-tests were used for statistical analyses. RESULTS: From aspirated bone marrow, multi-potent, heterogeneous BM-MSCs purified from hematopoietic stem cell contamination were obtained. FGF-2 significantly enhanced pig BM-MSC osteogenic differentiation and proliferation, with 5 ng/ml determined as the optimal dosage. Pig BM-MSCs integrated readily with Gelfoam and maintained viability and proliferative ability. After integration with Gelfoam scaffolds, 2.4-5.8×10(7 autologous BM-MSCs (undifferentiated or differentiated were transplanted to each experimental DO site. Among 8 evaluable DO sites included in the final analyses, the experimental DO sites demonstrated less interfragmentary mobility, more advanced gap obliteration, higher mineral content and faster mineral apposition than the control sites, and all transplanted scaffolds

  13. Data on bone marrow stem cells delivery using porous polymer scaffold

    Directory of Open Access Journals (Sweden)

    Ramasatyaveni Geesala

    2016-03-01

    Full Text Available Low bioavailability and/or survival at the injury site of transplanted stem cells necessitate its delivery using a biocompatible, biodegradable cell delivery vehicle. In this dataset, we report the application of a porous biocompatible, biodegradable polymer network that successfully delivers bone marrow stem cells (BMSCs at the wound site of a murine excisional splint wound model. In this data article, we are providing the additional data of the reference article “Porous polymer scaffold for on-site delivery of stem cells – protects from oxidative stress and potentiates wound tissue repair” (Ramasatyaveni et al., 2016 [1]. This data consists of the characterization of bone marrow stem cells (BMSCs showing the pluripotency and stem cell-specific surface markers. Image analysis of the cellular penetration into PEG–PU polymer network and the mechanism via enzymatic activation of MMP-2 and MMP-13 are reported. In addition, we provide a comparison of various routes of transplantation-mediated BMSCs engraftment in the murine model using bone marrow transplantation chimeras. Furthermore, we included in this dataset the engraftment of BMSCs expressing Sca-1+Lin−CD133+CD90.2+ in post-surgery day 10.

  14. Evaluation of Cartilage Repair by Mesenchymal Stem Cells Seeded on a PEOT/PBT Scaffold in an Osteochondral Defect

    NARCIS (Netherlands)

    Barron, V.; Merghani, K.; Shaw, G.; Coleman, C. M.; Hayes, J. S.; Ansboro, S.; Manian, A.; O’Malley, G.; Connolly, E.; Nandakumar, A.; van Blitterswijk, C. A.; Habibovic, P.; Moroni, L.; Shannon, F.; Murphy, J. M.; Barry, F.

    2015-01-01

    The main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scan

  15. Reduced inflammatory cell recruitment and tissue damage in spinal cord injury by acellular spinal cord scaffold seeded with mesenchymal stem cells.

    Science.gov (United States)

    Wang, Yu-Hai; Chen, Jian; Zhou, Jing; Nong, Feng; Lv, Jin-Han; Liu, Jia

    2017-01-01

    Therapy using acellular spinal cord (ASC) scaffolds seeded with bone marrow stromal cells (BMSCs) has previously been shown to restore function of the damaged spinal cord and improve functional recovery in a rat model of acute hemisected spinal cord injury (SCI). The aim of the present study was to determine whether BMSCs and ASC scaffolds promote the functional recovery of the damaged spinal cord in a rat SCI model through regulation of apoptosis and immune responses. Whether this strategy regulates secondary inflammation, which is characterized by the infiltration of immune cells and inflammatory mediators to the lesion site, in SCI repair was investigated. Basso, Beattie, and Bresnahan scores revealed that treatment with BMSCs seeded into an ASC scaffold led to a significant improvement in motor function recovery compared with treatment with an ASC scaffold alone or untreated controls at 2 and 8 weeks after surgery (Pscaffold significantly decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, as compared with the ASC scaffold only and control groups. These results suggested that the use of BMSCs decreased the apoptosis of neural cells and thereby limited tissue damage at the lesion site. Notably, the use of BMSCs with an ASC scaffold also decreased the recruitment of macrophages (microglia; Pscaffold only groups. BMSCs regulated inflammatory cell recruitment to promote functional recovery. However, there was no significant difference in IgM-positive expression among the three groups (P>0.05). The results of this study demonstrated that BMSCs seeded into ASC scaffolds for repair of spinal cord hemisection defects promoted functional recovery through the early regulation of inflammatory cell recruitment with inhibition of apoptosis and secondary inflammation.

  16. Research data supporting “Towards Cellular Sieving: Exploring the Limits of Scaffold Accessibility for Cell-Type Specific Invasion”

    OpenAIRE

    Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G.; Best, Serena M.; Cameron, Ruth E.

    2016-01-01

    Zip folder containing sample reconstructed Micro-CT scans (.tif files) from each scaffold condition. Image pixel size is 3.1 μm. Each image plane is perpendicular to the direction of cell invasion (i.e. parallel to the seeding plane). Two Microsoft Excel files, containing the raw measurements of pore size and of L and d (as defined in the manuscript) for calculation of percolation diameter. Units are in pixels unless specified. Zip folder containing raw images (.png files) of each scaffold se...

  17. Design of biomimetic and bioactive cold plasma-modified nanostructured scaffolds for enhanced osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Wang, Mian; Cheng, Xiaoqian; Zhu, Wei; Holmes, Benjamin; Keidar, Michael; Zhang, Lijie Grace

    2014-03-01

    The objective of this study was to design a biomimetic and bioactive tissue-engineered bone construct via a cold atmospheric plasma (CAP) treatment for directed osteogenic differentiation of human bone morrow mesenchymal stem cells (MSCs). Porous nanocrystalline hydroxyapatite/chitosan scaffolds were fabricated via a lyophilization procedure. The nanostructured bone scaffolds were then treated with CAP to create a more favorable surface for cell attachment, proliferation, and differentiation. The CAP-modified scaffolds were characterized via scanning electron microscope, Raman spectrometer, contact angle analyzer, and white light interferometer. In addition, optimal CAP treatment conditions were determined. Our in vitro study shows that MSC adhesion and infiltration were significantly enhanced on CAP modified scaffolds. More importantly, it was demonstrated that CAP-modified nanostructured bone constructs can greatly promote total protein, collagen synthesis, and calcium deposition after 3 weeks of culture, thus making them a promising implantable scaffold for bone regeneration. Moreover, the fibronectin and vitronection adsorption experiments by enzyme-linked immunosorbent assay demonstrated that more adhesion-mediated protein adsorption on the CAP-treated scaffolds. Since the initial specific protein absorption on scaffold surfaces can lead to further recruitment as well as activation of favorable cell functions, it is suggested that our enhanced stem cell growth and osteogenic function may be related to more protein adsorption resulting from surface roughness and wettability modification. The CAP modification method used in this study provides a quick one-step process for cell-favorable tissue-engineered scaffold architecture remodeling and surface property alteration.

  18. Glycerol-mediated nanostructure modification leading to improved transparency of porous polymeric scaffolds for high performance 3D cell imaging.

    Science.gov (United States)

    Zhao, Shan; Shen, Zhiyuan; Wang, Jingyu; Li, Xiaokang; Zeng, Yang; Wang, Bingjie; He, Yonghong; Du, Yanan

    2014-07-14

    Glycerol is among the most commonly used optical clearing agents for tissues clearance largely due to refractive index (RI) matching between glycerol and the submerged tissues. Here we applied glycerol as structure modifier at both macroscopic (as porogen) and nanoscopic (as nanostructure ameliorant) scales to fabricate transparent porous scaffolds made from poly(ethylene glycol) (PEG) as well as other widely used biomaterials (e.g., PLGA, PA, or gelatin), whose nanostructures, in the scale of light wavelength, dominantly improved the optical transmittance of the scaffolds even when immersed in RI mismatched medium (e.g., culture medium or water). We further exploited the clearing mechanisms based on Mie scattering theory, illustrating that conformational changes of polymer chains induced by solvent effects of glycerol enhanced the anisotropy (i.e., directional alignment) of the nanostructures, leading to reduced crystallinity and scattering of the resulted PEG scaffolds. Our findings represent the first and systematic demonstration with both experimental and theoretical evidence in effectively clearing porous polymeric scaffolds by mechanisms other than RI matching, which could tackle the limitations of current optical imaging of cells cultured within three-dimensional (3D) opaque porous scaffolds, such as poor visibility, low spatial resolution, and small penetration depth.

  19. Aerogel microspheres from natural cellulose nanofibrils and their application as cell culture scaffold.

    Science.gov (United States)

    Cai, Hongli; Sharma, Sudhir; Liu, Wenying; Mu, Wei; Liu, Wei; Zhang, Xiaodan; Deng, Yulin

    2014-07-14

    We demonstrated that ultralight pure natural aerogel microspheres can be fabricated using cellulose nanofibrials (CNF) directly. Experimentally, the CNF aqueous gel droplets, produced by spraying and atomizing through a steel nozzle, were collected into liquid nitrogen for instant freezing followed by freeze-drying. The aerogel microspheres are highly porous with bulk density as low as 0.0018 g cm(-3). The pore size of the cellulose aeogel microspheres ranges from nano- to macrometers. The unique ultralight and high porous structure ensured high moisture (~90 g g(-1)) and water uptake capacity (~100 g g(-1)) of the aerogel microspheres. Covalent cross-linking between the native nanofibrils and cross-linkers made the aerogel microspheres very stable even in a harsh environment. The present study also confirmed this kind of aerogel microspheres from native cellulose fibers can be used as cell culture scaffold.

  20. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

    Directory of Open Access Journals (Sweden)

    Eslahi N

    2013-11-01

    Full Text Available Neda Eslahi,1,2,* Mahmoud Reza Hadjighassem,1,3 Mohammad Taghi Joghataei,1,2 Tooba Mirzapour,4 Mehrdad Bakhtiyari,1,2 Malak Shakeri,5 Vahid Pirhajati,1,2 Peymaneh Shirinbayan,6,* Morteza Koruji1,21Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; 2Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 3Department of Neurosciences, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran; 5Department of Animal Science, Agricultural Campus, University of Tehran, Tehran, Iran; 6Pediatric Neuro-Rehabilitation Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran*These authors contributed equally to this articleIntroduction: A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs and testis tissues.Methods: The isolated spermatogonial cells were divided into six culture groups: (1 fresh spermatogonial cells, (2 fresh spermatogonial cells seeded onto PLLA, (3 frozen-thawed spermatogonial cells, (4 frozen-thawed spermatogonial cells seeded onto PLLA, (5 spermatogonial cells obtained from frozen-thawed testis tissue, and (6 spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFRα-1, PLZF, Mvh(VASA, Itgα6, and Itgβ1

  1. Poly(lactic-co-glycolic Acid/Nanohydroxyapatite Scaffold Containing Chitosan Microspheres with Adrenomedullin Delivery for Modulation Activity of Osteoblasts and Vascular Endothelial Cells

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    Lin Wang

    2013-01-01

    Full Text Available Adrenomedullin (ADM is a bioactive regulatory peptide that affects migration and proliferation of diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. This study investigated the effects of sustained release of ADM on the modulation activity of osteoblasts and vascular endothelial cells in vitro. Chitosan microspheres (CMs were developed for ADM delivery. Poly(lactic-co-glycolic acid and nano-hydroxyapatite were used to prepare scaffolds containing microspheres with ADM. The CMs showed rough surface morphology and high porosity, and they were well-distributed. The scaffolds exhibited relatively uniform pore sizes with interconnected pores. The addition of CMs improved the mechanical properties of the scaffolds without affecting their high porosity. In vitro degradation tests indicated that the addition of CMs increased the water absorption of the scaffolds and inhibited pH decline of phosphate-buffered saline medium. The expression levels of osteogenic-related and angiogenic-related genes were determined in MG63 cells and in human umbilical vein endothelial cells cultured on the scaffolds, respectively. The expression levels of osteogenic-related and angiogenic-related proteins were also detected by western blot analysis. Their expression levels in cells were improved on the ADM delivery scaffolds at a certain time point. The in vitro evaluation suggests that the microsphere-scaffold system is suitable as a model for bone tissue engineering.

  2. HPLC detection of loss rate and cell migration of HUVECs in a proanthocyanidin cross-linked recombinant human collagen-peptide (RHC)–chitosan scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing; Deng, Aipeng [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Yang [Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD (United Kingdom); Gao, Lihu; Xu, Na; Liu, Xin; Hu, Lunxiang; Chen, Junhua [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Yang, Shulin, E-mail: yshulin@njust.edu.cn [School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2015-11-01

    Porous scaffolds with appropriate pore structure, biocompatibility, mechanical property and processability play an important role in tissue engineering. In this paper, we fabricated a recombinant human collagen-peptide (RHC)–chitosan scaffold cross-linked by premixing 30% proanthocyanidin (PA) in one-step freeze-drying. To remove the residual acetic acid, optimized 0.2 M phosphate buffer of pH 6.24 with 30% ethanol (PBSE) was selected to neutralize the lyophilized scaffold followed by three times deionized water rinse. Ninhydrin assay was used to characterize the components loss during the fabrication process. To detect the exact RHC loss under optimized neutralization condition, high performance liquid chromatography (HPLC) equipped size exclusion chromatography column was used and the total RHC loss rate through PBSE rinse was 19.5 ± 5.08%. Fourier transform infrared spectroscopy (FT-IR) indicated hydrogen bonding among RHC, chitosan and PA, it also presented a probative but not strong hydrophobic interaction between phenyl rings of polyphenols and pyrrolidine rings of proline in RHC. Further, human umbilical vein endothelial cell (HUVEC) viability analyzed by a scanning electron microscope (SEM) and acridine orange/ethidium bromide (AO/EB) fluorescence staining exhibited that this scaffold could not only promote cell proliferation on scaffold surface but also permit cells migration into the scaffold. qRT-PCR exhibited that the optimized scaffold could stimulate angiogenesis associated genes VEGF and CD31 expression. These characterizations indicated that this scaffold can be considered as an ideal candidate for tissue engineering. - Highlights: • PA cross-linked recombinant human collagen–chitosan scaffold. • Fabrication in one-step lyophilization with neutralization. • HPLC detection of RHC loss rate • HUVEC proliferation and migration in scaffold • Angiogenesis associated gene expressions were increased in scaffold cell culturing.

  3. Micro- and Macrostructured PLGA/Gelatin Scaffolds Promote Early Cardiogenic Commitment of Human Mesenchymal Stem Cells In Vitro

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    Caterina Cristallini

    2016-01-01

    Full Text Available The biomaterial scaffold plays a key role in most tissue engineering strategies. Its surface properties, micropatterning, degradation, and mechanical features affect not only the generation of the tissue construct in vitro, but also its in vivo functionality. The area of myocardial tissue engineering still faces significant difficulties and challenges in the design of bioactive scaffolds, which allow composition variation to accommodate divergence in the evolving myocardial structure. Here we aimed at verifying if a microstructured bioartificial scaffold alone can provoke an effect on stem cell behavior. To this purpose, we fabricated microstructured bioartificial polymeric constructs made of PLGA/gelatin mimicking anisotropic structure and mechanical properties of the myocardium. We found that PLGA/gelatin scaffolds promoted adhesion, elongation, ordered disposition, and early myocardial commitment of human mesenchymal stem cells suggesting that these constructs are able to crosstalk with stem cells in a precise and controlled manner. At the same time, the biomaterial degradation kinetics renders the PLGA/gelatin constructs very attractive for myocardial regeneration approaches.

  4. Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds

    NARCIS (Netherlands)

    Leferink, Anne M; Chng, Yhee-Cheng; van Blitterswijk, Clemens A; Moroni, Lorenzo

    2015-01-01

    One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with d

  5. Distribution and viability of fetal and adult human bone marrow stromal cells in a biaxial rotating vessel bioreactor after seeding on polymeric 3D additive manufactured scaffolds

    NARCIS (Netherlands)

    Leferink, Anne M.; Chng, Yhee-Cheng; Blitterswijk, van Clemens A.; Moroni, Lorenzo

    2015-01-01

    One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with d

  6. Constructive tissue remodeling of biologic scaffolds: A phenomenon associated with scaffold characteristics and distinctive macrophage phenotypes

    Science.gov (United States)

    Brown, Bryan Nicklaus

    Scaffolds composed of extracellular matrix (ECM) have been shown to promote formation of site-specific, functional host tissue following implantation in a number of preclinical and clinical settings. However, the exact mechanisms by which ECM scaffolds are able to promote this type of "constructive tissue remodeling" are unknown. Further, the ability of ECM scaffolds to promote constructive tissue remodeling appears to be dependent on the methods used in their production and the applications in which they are utilized. Therefore, a comprehensive understanding of ECM scaffold characteristics and their effects upon the host response and subsequent tissue remodeling outcome is essential to the design of intelligent scaffolds for specific clinical applications. The present work investigated the effects of tissue source and chemical cross-linking upon the resulting ECM scaffolds, showing that ECM scaffold materials have distinct ultrastructural and compositional characteristics which are dependant on the anatomic location from which the scaffolds are derived and the methods used in their production. These characteristics were associated with distinct patterns of cell behavior in vitro. Distinct tissue remodeling outcomes were observed following implantation of a subset of these scaffold materials in a rat abdominal wall musculature reconstruction model. Acellular, non-cross-linked ECM was associated with constructive tissue remodeling while scaffolds that contained cellular components or were chemically cross-linked resulted in dense connective tissue deposition or encapsulation, respectively. Despite differences in the tissue remodeling outcome, a histologically similar population of macrophages was observed following implantation in each of these cases. Therefore, the phenotype of the macrophage population participating in the host response was investigated. It was shown that scaffolds which resulted in constructive tissue remodeling were associated with an increase

  7. Quantitative estimates of vascularity in a collagen-based cell scaffold containing basic fibroblast growth factor by non-invasive near-infrared spectroscopy for regenerative medicine

    Science.gov (United States)

    Kushibiki, Toshihiro; Awazu, Kunio

    2008-04-01

    Successful tissue regeneration required both cells with high proliferative and differentiation potential and an environment permissive for regeneration. These conditions can be achieved by providing cell scaffolds and growth factors that induce angiogenesis and cell proliferation. Angiogenenis within cell scaffolds is typically determined by histological examination with immunohistochemical markers for endothelium. Unfortunately, this approach requires removal of tissue and the scaffold. In this study, we examined the hemoglobin content of implanted collagen-based cell scaffolds containing basic fibroblast growth factor (bFGF) in vivo by non-invasive near infrared spectroscopy (NIRS). We also compared the hemoglobin levels measured by NIRS to the hemoglobin content measured with a conventional biological assay. Non-invasive NIRS recordings were performed with a custom-built near-infrared spectrometer using light guide-coupled reflectance measurements. NIRS recordings revealed that absorbance increased after implantation of collagen scaffolds containing bFGF. This result correlated (R2=0.93) with our subsequent conventional hemoglobin assay. The NIRS technique provides a non-invasive method for measuring the degree of vascularization in cell scaffolds. This technique may be advantageous for monitoring angiogenesis within different cell scaffolds, a prerequisite for effective tissue regeneration.

  8. In vitro mineralization of MC3T3-E1 osteoblast-like cells on collagen/nano-hydroxyapatite scaffolds coated carbon/carbon composites.

    Science.gov (United States)

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

    2016-02-01

    Collagen/nano-hydroxyapatite (collagen/nHA) scaffolds were successfully prepared on carbon/carbon composites as bioactive films using the layer-by-layer coating method. Surface characterizations of collagen/nHA scaffolds were detected by scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Compressive strengths of the scaffolds were evaluated by a universal test machine. In vitro biological performances were determined using scaffolds seeded with MC3T3-E1 osteoblasts-like cells and cultured in mineralization medium for up to 21 days. In addition, cellular morphologies and several related gene expressions of MC3T3-E1 cells in the scaffolds were also evaluated. Chemical and morphological analysis showed that the scaffolds had uniform pore sizes and unified phase composition. Mechanical testing indicated that the collagen/nHA scaffolds had the highest compressive strength in 50% of strain condition when the proportion of collagen and nano-hydroxyapatite was 1:3. Cellular morphology observations and cytology tests indicated that MC3T3-E1 cells were adhered on these scaffolds and proliferated. SEM photographs and gene expressions showed that mineralized MC3T3-E1 cells and newly formed extra cellular matrix (ECM) filled up the pores of the scaffolds after the 3-week mineralization inducement. Nano-sized apatite particles were secreted from MC3T3-E1 cells and combined with the reconstructed ECM. Collectively, collagen/nHA scaffolds provided C/C composites with a biomimetic surface for cell adhesion, proliferation and mineralized extra cellular matrices formation.

  9. Analysis of production of the decellularized scaffolds and their potential use in cardiovascular surgery

    Directory of Open Access Journals (Sweden)

    Brumberg V.A.

    2015-12-01

    Full Text Available Data on application of decellularized scaffolds and tissue-engineered vascular conduits in the field of cardiovascular surgery have been analyzed, and also techniques for their procurement have been studied. For finding, selection and synthesis resources of research data from the systems Pubmed, ScienceDirect were used. The preference was given to the most informative, comprehensive and contemporary publications.

  10. Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chih-Hao [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Lee, Ming-Yih [Graduate Institute of Medical Mechatronics, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Shyu, Victor Bong-Hang; Chen, Yi-Chieh; Chen, Chien-Tzung [Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Chen, Jyh-Ping, E-mail: jpchen@mail.cgu.edu.tw [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan 333, Taiwan, ROC (China)

    2014-07-01

    Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction. - Highlights: • Selective laser sintered polycaprolactone scaffolds are prepared. • Scaffolds are surface modified through immersion coating with gelatin or collagen. • Collagen-scaffold is the best for cartilage tissue engineering in vitro. • Chondrocytes/collagen-scaffold reveals enhanced cartilage tissue formation in vivo.

  11. Nanostructured porous silicon: The winding road from photonics to cell scaffolds. A review.

    Directory of Open Access Journals (Sweden)

    Jacobo eHernandez-Montelongo

    2015-05-01

    Full Text Available For over 20 years nanostructured porous silicon (nanoPS has found a vast number of applications in the broad fields of photonics and optoelectronics, triggered by the discovery of its photoluminescent behavior in 1990. Besides, its biocompatibility, biodegradability, and bioresorbability make porous silicon (PSi an appealing biomaterial. These properties are largely a consequence of its particular susceptibility to oxidation, leading to the formation of silicon oxide which is readily dissolved by body fluids. This paper reviews the evolution of the applications of PSi and nanoPS from photonics through biophotonics, to their use as cell scaffolds, whether as an implantable substitute biomaterial, mainly for bony and ophthalmological tissues, or as an in-vitro cell conditioning support, especially for pluripotent cells. For any of these applications, PSi/nanoPS can be used directly after synthesis from Si wafers, upon appropriate surface modification processes, or as a composite biomaterial. Unedited studies of fluorescently active PSi structures for cell culture are brought to evidence the margin for new developments.

  12. Using Polymer Confinement for Stem Cell Differentiation: 3D Printed vs Molded Scaffolds

    Science.gov (United States)

    Rafailovich, Miriam

    Additive manufacturing technologies are increasingly being used to replace standard extrusion or molding methods in engineering polymeric biomedical implants, which can be further seeded with cells for tissue regeneration. The principal advantage of this new technology is the ability to print directly from a scan and hence produce parts which are an ideal fit for an individual, eliminating much of the sizing and fitting associated with standard manufacturing methods. The question though arises whether devices which may be macroscopically similar, serve identical functions and are produced from the same material, interact in the same manner with cells and living tissue. Here we show that fundamental differences can exist between 3-D printed and extruded scaffolds which can impact stem cell differentiation and lineage selection. We will show how polymer confinement inherent in these methods affect the printed features on multiple length scales. We will also and how the differentiation of stem cells is affected by substrate heterogeneity in both morphological and mechanical features. NSF-Inspire award # 1344267.

  13. Nanostructured Porous Silicon: The Winding Road from Photonics to Cell Scaffolds – A Review

    Science.gov (United States)

    Hernández-Montelongo, Jacobo; Muñoz-Noval, Alvaro; García-Ruíz, Josefa Predestinación; Torres-Costa, Vicente; Martín-Palma, Raul J.; Manso-Silván, Miguel

    2015-01-01

    For over 20 years, nanostructured porous silicon (nanoPS) has found a vast number of applications in the broad fields of photonics and optoelectronics, triggered by the discovery of its photoluminescent behavior in 1990. Besides, its biocompatibility, biodegradability, and bioresorbability make porous silicon (PSi) an appealing biomaterial. These properties are largely a consequence of its particular susceptibility to oxidation, leading to the formation of silicon oxide, which is readily dissolved by body fluids. This paper reviews the evolution of the applications of PSi and nanoPS from photonics through biophotonics, to their use as cell scaffolds, whether as an implantable substitute biomaterial, mainly for bony and ophthalmological tissues, or as an in vitro cell conditioning support, especially for pluripotent cells. For any of these applications, PSi/nanoPS can be used directly after synthesis from Si wafers, upon appropriate surface modification processes, or as a composite biomaterial. Unedited studies of fluorescently active PSi structures for cell culture are brought to evidence the margin for new developments. PMID:26029688

  14. Design and Validation of a Cyclic Strain Bioreactor to Condition Spatially-Selective Scaffolds in Dual Strain Regimes

    Directory of Open Access Journals (Sweden)

    J. Matthew Goodhart

    2014-03-01

    Full Text Available The objective of this study was to design and validate a unique bioreactor design for applying spatially selective, linear, cyclic strain to degradable and non-degradable polymeric fabric scaffolds. This system uses a novel three-clamp design to apply cyclic strain via a computer controlled linear actuator to a specified zone of a scaffold while isolating the remainder of the scaffold from strain. Image analysis of polyethylene terephthalate (PET woven scaffolds subjected to a 3% mechanical stretch demonstrated that the stretched portion of the scaffold experienced 2.97% ± 0.13% strain (mean ± standard deviation while the unstretched portion experienced 0.02% ± 0.18% strain. NIH-3T3 fibroblast cells were cultured on the PET scaffolds and half of each scaffold was stretched 5% at 0.5 Hz for one hour per day for 14 days in the bioreactor. Cells were checked for viability and proliferation at the end of the 14 day period and levels of glycosaminoglycan (GAG and collagen (hydroxyproline were measured as indicators of extracellular matrix production. Scaffolds in the bioreactor showed a seven-fold increase in cell number over scaffolds cultured statically in tissue culture plastic petri dishes (control. Bioreactor scaffolds showed a lower concentration of GAG deposition per cell as compared to the control scaffolds largely due to the great increase in cell number. A 75% increase in hydroxyproline concentration per cell was seen in the bioreactor stretched scaffolds as compared to the control scaffolds. Surprisingly, little differences were experienced between the stretched and unstretched portions of the scaffolds for this study. This was largely attributed to the conditioned and shared media effect. Results indicate that the bioreactor system is capable of applying spatially-selective, linear, cyclic strain to cells growing on polymeric fabric scaffolds and evaluating the cellular and matrix responses to the applied strains.

  15. Design and characterization of core-shell mPEG-PLGA composite microparticles for development of cell-scaffold constructs.

    Science.gov (United States)

    Wen, Yanhong; Gallego, Monica Ramos; Nielsen, Lene Feldskov; Jorgensen, Lene; Møller, Eva Horn; Nielsen, Hanne Mørck

    2013-09-01

    Appropriate scaffolds capable of providing suitable biological and structural guidance are of great importance to generate cell-scaffold constructs for cell-based tissue engineering. The aim of the present study was to develop composite microparticles with a structure to provide functionality as a combined drug delivery/scaffold system. Composite microparticles were produced by incorporating either alginate/dermatan sulfate (Alg/DS) or alginate/chitosan/dermatan sulfate (Alg/CS/DS) particles in mPEG-PLGA microparticles using coaxial ultrasonic atomization. The encapsulation and distribution of Alg/DS or Alg/CS/DS particles in the mPEG-PLGA microparticles were significantly dependent on the operating conditions, including the flow rate ratio (Qout/Qin) and the viscosity of the polymer solutions (Vout, Vin) between the outer and the inner feeding channels. The core-shell composite microparticles containing the Alg/DS particles or the Alg/CS/DS particles displayed 40% and 65% DS release in 10 days, respectively, as compared to the DS directly loaded microparticles showing 90% DS release during the same time interval. The release profiles of DS correlate with the cell proliferation of fibroblasts, i.e. more sustainable cell growth was induced by the DS released from the core-shell composite microparticles comprising Alg/CS/DS particles. After seeding fibroblasts onto the composite microparticles, excellent cell adhesion was observed, and a successful assembly of the cell-scaffold constructs was induced within 7 days. Therefore, the present study demonstrates a novel strategy for fabrication of core-shell composite microparticles comprising additional particulate drug carriers in the core, which provides controlled delivery of DS and favorable cell biocompatibility; an approach to potentially achieve cell-based tissue regeneration.

  16. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Directory of Open Access Journals (Sweden)

    Sunita Sharma

    Full Text Available Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC. This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2 in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone{poly(LLA-co-CL}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2 and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  17. Combining 3-dimensional degradable electrostatic spinning scaffold and dental follicle cells to build peri-implant periodontium

    Directory of Open Access Journals (Sweden)

    Ximu Zhang

    2013-01-01

    Full Text Available Introduction: Some inevitable problems, such as concentrated bite force and lacked ability of self-renewal, are proved to be the major challenge in the management of implants failures. Thus, it is meaningful to find an ideal dental implant harboring its own peri-implant periodontium, just as the natural teeth. Various studies attempted to reconstruct the periodontium around implants, but unfortunately, it was previously revealed that the artificial periodotium around implants was just a wilderness of fibers, while without the physiological function of natural periodontium, like sensory and homeostatic. The Hypothesis: In this paper, we propose a hypothesis that a modified three-dimensional scaffold with reconstructed peri-implant tissues can be a network for stem cells differentiation. After seeded on the scaffold, stem cells produce various growth factors and differentiate to different orientations in places necessary. This hypothesis, if proven to be valid, will offer a novel and effective therapy for the restoration of missing teeth by implant. Evaluation of the Hypothesis: The scaffold involves three different tissues. Though degradation rate of electrospinning scaffold is under control, its degradation rate should be in consistent with the generation of three tissues. Therefore, the relative experiments are necessary to define the best rate of degradation. Further verification is necessary to check whether the rebuilt cementum, bone and periodontium are strong enough to keep the implant stable and maintain its function.

  18. Manipulation of chemical composition and architecture of non-biodegradable poly(ethylene terephthalate)/chitosan fibrous scaffolds and their effects on L929 cell behavior

    Energy Technology Data Exchange (ETDEWEB)

    Veleirinho, Beatriz [QOPNA Research Unit, Department of Chemistry, University of Aveiro, 3810-193 Aveiro (Portugal); Berti, Fernanda V. [Integrated Technologies Laboratory, Chemical and Food Engineering Department, Federal University of Santa Catarina, 88040-900 Florianopolis (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianopolis (Brazil); Maraschin, Marcelo [Department of Plant Science, Federal University of Santa Catarina, 88040-900 Florianopolis (Brazil); Ribeiro-do-Valle, Rosa M. [Department of Pharmacology, Federal University of Santa Catarina, 88040-900 Florianopolis (Brazil); Lopes-da-Silva, Jose A., E-mail: jals@ua.pt [QOPNA Research Unit, Department of Chemistry, University of Aveiro, 3810-193 Aveiro (Portugal)

    2013-01-01

    Microporous, non-woven fibrous scaffolds made of poly(ethylene terephthalate) and chitosan were produced by electrospinning. Fiber morphology, diameter, pore size, and wettability were manipulated by varying the chemical composition of the electrospinning solution, i.e. chitosan concentration and molecular weight, and by post-electrospinning treatment with glutaraldehyde. In vitro studies were conducted using a fibroblast cell line toward a comprehensive understanding of how scaffolds characteristics can modulate the cell behavior, i.e. viability, adhesion, proliferation, extracellular matrix secretion, and three-dimensional colonization. Substantial differences were found as a result of scaffold morphological changes. Higher levels of adhesion, spreading, and superficial proliferation were achieved for scaffolds with smaller fiber and pore diameters while cell penetration and internal colonization were enhanced for scaffolds with larger pores. Additionally, the available area for cell adhesion, which is related to fiber and pore size, was a crucial factor for the viability of L929 cells. This paper provides significant insights for the development and optimization of electrospun scaffolds toward an improved biological performance. Highlights: Black-Right-Pointing-Pointer Hybrid PET/chitosan mats were produced by electrospinning. Black-Right-Pointing-Pointer Scaffold architecture was manipulated by changing composition of the spun solution. Black-Right-Pointing-Pointer The scaffolds showed in vitro biocompatibility to L929 cells. Black-Right-Pointing-Pointer Smaller fiber diameters and pore areas allowed for higher levels of cell adhesion and proliferation. Black-Right-Pointing-Pointer A 3D cell colonization was achieved for scaffolds with higher fiber diameters.

  19. Tooth root regeneration using dental follicle cell sheets in combination with a dentin matrix - based scaffold.

    Science.gov (United States)

    Yang, Bo; Chen, Gang; Li, Jie; Zou, Qing; Xie, Dan; Chen, Yali; Wang, Hang; Zheng, Xiaohui; Long, Jie; Tang, Wei; Guo, Weihua; Tian, Weidong

    2012-03-01

    Stem cell mediated tissue engineering has been acknowledged as a prospective strategy for repairing and replacing damaged and lost tissues. However, the low survival rate of implanted stem cells proves to be a major challenge in the management of transplantation failures. While previous studies have indicated the effectiveness of tissue engineered cell sheets in improving the survival rate of implanted cells, we have recently demonstrated the use of treated dentin matrix (TDM) as a biological scaffold and dental follicle cells (DFCs) as the seeding cells for dentinogenesis and tooth root construction. This study proposes a strategy utilizing TDM with human dental follicle cell sheets (DFCSs) for root regeneration. The biological characteristics and changes of human DFCSs under the effect of TDM were studied with scanning electron microscopy, transmission electron microscopy, immunofluorescence microscopy, immunohistochemistry and quantitative real-time PCR. DFCSs combined with TDM were implanted subcutaneously into the dorsum of mice. Histological examination of the harvested grafts revealed a whirlpool-like alignment of the DFCs in multiple layers that were positive for COLI, integrinβ1, fibronectin and alkaline phosphatase (ALP), suggestive of the formation of a rich extracellular matrix. DFCSs, under the effect of TDM, highly expressed DMP-1 and bone sialoprotein (BSP), indicating their potential for odontogenesis and osteogenesis. Importantly, in vivo, TDM could induce and support DFCSs to develop new dentin-pulp like tissues and cementum-periodontal complexes that were positive for markers such as DSP, nestin and VIII factors, COLI and cementum attachment protein (CAP), implying successful root regeneration. Therefore, DFCSs combined with TDM may prove to be a better strategy for the construction of tooth root, and is a prospective approach that could be utilized for the treatment of root or tooth defect or loss in future.

  20. Biocompatibility evaluation of electrospun aligned poly(propylene carbonate) nanofibrous scaffolds with peripheral nerve tissues and cells in vitro

    Institute of Scientific and Technical Information of China (English)

    WANG Yu; ZHAO Zhe; ZHAO Bin; QI Hong-xu; PENG Jiang; ZHANG Li; XU Wen-jing; HU Ping; LU Shi-bi

    2011-01-01

    Background Peripheral nerve regeneration across large gaps is clinically challenging. Scaffold design plays a pivotal role in nerve tissue engineering. Recently, nanofibrous scaffolds have proven a suitable environment for cell attachment and proliferation due to similarities of their physical properties to natural extracellular matrix. Poly(propylene carbonate)(PPC) nanofibrous scaffolds have been investigated for vascular tissue engineering. However, no reports exist of PPC nanofibrous scaffolds for nerve tissue engineering. This study aimed to evaluate the potential role of aligned and random PPC nanofibrous scaffolds as substrates for peripheral nerve tissue and cells in nerve tissue engineering. Methods Aligned and random PPC nanofibrous scaffolds were fabricated by electrospinning and their chemical characterization were carried out using scanning electron microscopy (SEM). Dorsal root ganglia (DRG) from Sprague-Dawley rats were cultured on the nanofibrous substrates for 7 days. Neurite outgrowth and Schwann-cell migration from DRG were observed and quantified using immunocytochemistry and SEM. Schwann cells derived from rat sciatic nerves were cultured in electrospun PPC scaffold-extract fluid for 24, 48, 72 hours and 7 days. The viability of Schwann cells was evaluated by 3-[4,5-dimethyl(thiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT) assay. Results The diameter of aligned and random fibers ranged between 800 nm and 1200 nm, and the thickness of the films was approximately 10-20 μm. Quantification of aligned fiber films revealed approximately 90% alignment of all fibers along the longitudinal axis. However, with random fiber films, the alignment of fibers was random through all angle bins. Rat DRG explants were grown on PPC nanofiber films for up to 1 week. On the aligned fiber films, the majority of neurite outgrowth and Schwann cell migration from the DRG extended unidirectionally, parallel to the aligned fibers.However, on the random fiber films

  1. Hydrogels as feeder-free scaffolds for long-term self-renewal of mouse induced pluripotent stem (mips) cells.

    OpenAIRE

    2012-01-01

    Expanding undifferentiated induced pluripotent stem (iPS) cells in vitro is a basic requirement for application of iPS cells in both fundamental research and clinical regeneration. In this study, we intended to establish a simple, low cost and efficient method for the long-term self-renewal of mouse induced pluripotent stem (miPS) cells without using feeder-cells and adhesive proteins. Three scaffolds were selected for the long-term subculture of miPS cells over two months starting from passa...

  2. A natural product inspired hybrid approach towards the synthesis of novel pentamidine based scaffolds as potential anti-parasitic agents.

    Science.gov (United States)

    Tyagi, Vikas; Khan, Shahnawaz; Shivahare, Rahul; Srivastava, Khushboo; Gupta, Suman; Kidwai, Saqib; Srivastava, Kumkum; Puri, S K; Chauhan, Prem M S

    2013-01-01

    A natural product inspired molecular hybridization approach led us to a series of novel pentamidine based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial potential. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine). Additionally, anti-malarial screening of these compounds was also done and four compounds have shown superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.

  3. A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

    Science.gov (United States)

    2013-10-01

    biopolymer scaffold within the internal auditory meatus (IAM) of the guinea pig. (A) The lateral wall of an intact guinea pig temporal bone is shown......Nanofibrous Scaffold for Auditory Nerve Replacement 5b. GRANT NUMBER W81XWH-12-1-0492 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Betty Diamond

  4. Repairing rabbit radial defects by combining bone marrow stroma stem cells with bone scaffold material comprising a core-cladding structure.

    Science.gov (United States)

    Wu, H; Liu, G H; Wu, Q; Yu, B

    2015-10-05

    We prepared a bone scaffold material comprising a PLGA/β-TCP core and a Type I collagen cladding, and recombined it with bone marrow stroma stem cells (BMSCs) to evaluate its potential for use in bone tissue engineering by in vivo and in vitro experiments. PLGA/β-TCP without a cladding was used for comparison. The adherence rate of the BMSCs to the scaffold was determined by cell counting. Cell proliferation rate was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The osteogenic capability was evaluated by alkaline phosphatase activity. The scaffold materials were recombined with the BMSCs and implanted into a large segmental rabbit radial defect model to evaluate defect repair. Osteogenesis was assessed in the scaffold materials by histological and double immunofluorescence labeling, etc. The adherence number, proliferation number, and alkaline phosphatase expression of the cells on the bone scaffold material with core-cladding structure were significantly higher than the corresponding values in the PLGA/β-TCP composite scaffold material (P cladding structure completely degraded at the bone defect site and bone formation was completed. The rabbit large sentimental radial defect was successfully repaired. The degradation and osteogenesis rates matched well. The bone scaffold with core-cladding structure exhibited better osteogenic activity and capacity to repair a large segmental bone defect compared to the PLGA/β-TCP composite scaffold. The bone scaffold with core-cladding structure has excellent physical properties and biocompatibility. It is an ideal scaffold material for bone tissue engineering.

  5. Zeolite inorganic scaffolds for novel biomedical application: Effect of physicochemical characteristic of zeolite membranes on cell adhesion and viability

    Science.gov (United States)

    Tavolaro, Palmira; Catalano, Silvia; Martino, Guglielmo; Tavolaro, Adalgisa

    2016-09-01

    The design, preparation and selection of inorganic materials useful as functional scaffolds for cell adhesion is a complex question based both on the understanding of the chemical behavior of the materials and individual cells, and on their interactions. Pure zeolite membranes formed from synthetic crystals offer chemically-capable being modulated silanolic surfaces that are amenable to adhesion and growth of fibroblasts. We report the facile preparation of reusable, very longlasting, biocompatible, easily sterilized synthetic scaffolds in a zeolite membrane configuration, which are very stable in aqueous media (apart from ionic strength and pH values), able to adsorb pollutant species and to confine undesired toxic ions (present in culture media). This may ultimately lead to the development of cell supports for economic antibiotic-free culture media.

  6. Design, fabrication and perivascular implantation of bioactive scaffolds engineered with human adventitial progenitor cells for stimulation of arteriogenesis in peripheral ischemia.

    Science.gov (United States)

    Carrabba, M; De Maria, C; Oikawa, A; Reni, C; Rodriguez-Arabaolaza, I; Spencer, H; Slater, S; Avolio, E; Dang, Z; Spinetti, G; Madeddu, P; Vozzi, G

    2016-03-24

    Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.

  7. Scaffold characterization using NLO multimodal microscopy in metrology for regenerative medicine

    Science.gov (United States)

    Mortati, Leonardo; Divieto, Carla; Boffitto, Monica; Sartori, Susanna; Ciardelli, Gianluca; Sassi, Maria Paola

    2013-09-01

    Metrology in regenerative medicine aims to develop traceable measurement technologies for characterizing cellular and macromolecule behaviour in regenerative medicine products and processes. One key component in regenerative medicine is using three-dimensional porous scaffolds to guide cells during the regeneration process. The regeneration of specific tissues guided by tissue analogous substrates is dependent on diverse scaffold architectural properties that can be derived quantitatively from scaffolds images. This paper discuss the results obtained with the multimodal NLO microscope recently realized in our laboratory in characterizing 3D tissue engineered (TE) scaffolds colonized from human Mesenchimal stem cells (hMSC), focusing on the study of the three-dimensional metrological parameters.

  8. No Immunogenicity of IPS Cells in Syngeneic Host Studied by In Vivo Injection and 3D Scaffold Experiments

    Directory of Open Access Journals (Sweden)

    Suganya Thanasegaran

    2013-01-01

    Full Text Available Induced Pluripotent Stem Cells (IPSCs open the great possibility to employ patient’s own tissue to the previously incurable diseases. However these cells can be used in cell therapy only if they are not rejected when transplanted back into the syngeneic host. We found that the injection of iPSCs derived from different ages of mice into syngeneic C57BL/6 mice produced teratoma and was not rejected. Then we cultured iPSCs and myeloid differentiated iPSCs in three-dimensional porous scaffold and transplanted to C57BL/6 mice and BALB/C mice. After transplantation, we could observe the cell density inside the scaffold increased rapidly in syngeneic mice compared to the allogeneic mice indicating the favorable conditions supporting the growth of iPSCs in vivo. Unlike the allogeneic counterpart, we could not observe few infiltrating T cells inside the scaffold of syngeneic mice. These results contribute to the optimistic view of iPSCs for regenerative medicine in near future.

  9. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage.

    Science.gov (United States)

    Puvaneswary, Subramaniam; Raghavendran, Hanumantharao Balaji; Talebian, Sepehr; Murali, Malliga Raman; A Mahmod, Suhaeb; Singh, Simmrat; Kamarul, Tunku

    2016-04-12

    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.

  10. Bio-safe processing of polylactic-co-caprolactone and polylactic acid blends to fabricate fibrous porous scaffolds for in vitro mesenchymal stem cells adhesion and proliferation.

    Science.gov (United States)

    Salerno, Aurelio; Guarino, Vincenzo; Oliviero, Olimpia; Ambrosio, Luigi; Domingo, Concepción

    2016-06-01

    In this study, the design and fabrication of porous scaffolds, made of blends of polylactic-co-caprolactone (PLC) and polylactic acid (PLA) polymers, for tissue engineering applications is reported. The scaffolds are prepared by means of a bio-safe thermally induced phase separation (TIPS) approach with or without the addition of NaCl particles used as particulate porogen. The scaffolds are characterized to assess their crystalline structure, morphology and mechanical properties, and the texture of the pores and the pore size distribution. Moreover, in vitro human mesenchymal stem cells (hMSCs) culture tests have been carried out to demonstrate the biocompatibility of the scaffolds. The results of this study demonstrate that all of the scaffold materials processed by means of TIPS process are semi-crystalline. Furthermore, the blend composition affected polymer crystallization and, in turn, the nano and macro-structural properties of the scaffolds. Indeed, neat PLC and neat PLA crystallize into globular and randomly arranged sub micro-size scale fibrous conformations, respectively. Concomitantly, the addition of NaCl particles during the fabrication route allows for the creation of an interconnected network of large pores inside the primary structure while resulted in a significant decrease of scaffolds mechanical response. Finally, the results of cell culture tests demonstrate that both the micro and macro-structure of the scaffold affect the in vitro hMSCs adhesion and proliferation.

  11. Growth of Bone Marrow Derived Osteoblast-Like Cells into Coral Implant Scaffold: Preliminary Study on Malaysian Coral

    Directory of Open Access Journals (Sweden)

    K. A. AL-Salihi

    2009-01-01

    Full Text Available Problem statement: Biomaterial fabrication in Malaysia started as a consequence of the demand for cheaper implant materials. Various biomaterials have been developed utilizing local resources like Malaysian coral. Locally processed Malaysian coral need to be complemented with proper evaluation and testing including toxicology, biocompatibility, mechanical properties, physicochemical characterization and in vivo testing. The present study was carried out to assess natural coral of porites species as scaffold combined with in vitro expanded Bone Marrow Derived Osteoblast-Like cells (BM-DOL, in order to develop a tissue-engineered bone graft in a rat model. Approach: Coral was used in a block shape with a dimension of 10 mm length × 5 mm width × 5 mm thickness. BM-DOL cells were seeded into porous coral scaffold in a density of 5×106 mL-1. After 7 days of in vitro incubation in osteogenic medium, one block was processed for light (LM and Scanning Electron Microscopy (SEM observations while the other blocks were implanted subcutaneously in the back of 5 weeks-old Sprague-Dawely rats for 3 months. Coral blocks without cells were implanted as a control. The implants harvested and processed for gross inspection, histological and scanning electron microscopy observations. Results: Both LM and SEM showed attachment of well arrangement multilayer cells inside the pores of in vitro seeded coral scaffolds. Gross inspection of all in vivo coral-cell complexes implants revealed vascularized like bone tissue formation. Histological sections revealed mature bone formation occurred in the manner resemble intramembraneous bone formation. SEM observations revealed multi-layer cellular proliferation with abundant collagen covered the surface of coral implants. Control group showed resorbed coral block. Conclusion: This study demonstrated that Malaysian coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal

  12. Enhancement of cell-based therapeutic angiogenesis using a novel type of injectable scaffolds of hydroxyapatite-polymer nanocomposite microspheres.

    Directory of Open Access Journals (Sweden)

    Yohei Mima

    Full Text Available BACKGROUND: Clinical trials demonstrate the effectiveness of cell-based therapeutic angiogenesis in patients with severe ischemic diseases; however, their success remains limited. Maintaining transplanted cells in place are expected to augment the cell-based therapeutic angiogenesis. We have reported that nano-hydroxyapatite (HAp coating on medical devices shows marked cell adhesiveness. Using this nanotechnology, HAp-coated poly(l-lactic acid (PLLA microspheres, named nano-scaffold (NS, were generated as a non-biological, biodegradable and injectable cell scaffold. We investigate the effectiveness of NS on cell-based therapeutic angiogenesis. METHODS AND RESULTS: Bone marrow mononuclear cells (BMNC and NS or control PLLA microspheres (LA were intramuscularly co-implanted into mice ischemic hindlimbs. When BMNC derived from enhanced green fluorescent protein (EGFP-transgenic mice were injected into ischemic muscle, the muscle GFP level in NS+BMNC group was approximate fivefold higher than that in BMNC or LA+BMNC groups seven days after operation. Kaplan-Meier analysis demonstrated that NS+BMNC markedly prevented hindlimb necrosis (P<0.05 vs. BMNC or LA+BMNC. NS+BMNC revealed much higher induction of angiogenesis in ischemic tissues and collateral blood flow confirmed by three-dimensional computed tomography angiography than those of BMNC or LA+BMNC groups. NS-enhanced therapeutic angiogenesis and arteriogenesis showed good correlations with increased intramuscular levels of vascular endothelial growth factor and fibroblast growth factor-2. NS co-implantation also prevented apoptotic cell death of transplanted cells, resulting in prolonged cell retention. CONCLUSION: A novel and feasible injectable cell scaffold potentiates cell-based therapeutic angiogenesis, which could be extremely useful for the treatment of severe ischemic disorders.

  13. Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

    Institute of Scientific and Technical Information of China (English)

    YOU Hongbo; CHEN Anmin; SUN Kai; LIU Tie

    2011-01-01

    The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells(PSCs)into chondrocytes was studied.KLD-12 was synthesized by solid-state method.After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold,DNA release ability was investigated.PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold,and MTT assay was performed to investigate the cell proliferation,and ELASA assay was used to investigate the expression of TGF-β3.Specific cartilage matrix was examined by quantitative real-time PCR,immunohistochemistry and Alcian Blue staining.Compared with control group,DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,and maintained this high level within 2 weeks.MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group(P<0.05).Quantitative realtime PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group(P<0.01).ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group's PSCs,reached the peak after 72 h and then declined a little to the plateau phase.Compared with the control group,the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01).The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,which provided a flesh approach to cartilage tissue engineering.

  14. 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.

  15. Repetitive Gly-Leu-Lys-Gly-Glu-Asn-Arg-Gly-Asp peptide derived from collagen and fibronectin for improving cell-scaffold interaction.

    Science.gov (United States)

    Chaisri, Patcharaporn; Chingsungnoen, Artit; Siri, Sineenat

    2015-03-01

    Suitable scaffolds for tissue engineering should provide a microenvironment for cell dwelling and directing cell behavior that resemble the native environment. Three-dimensional geometry of electrospun scaffolds well supports cell deposition, but they often lack biomacromolecules to induce cell responses. In this work, the repetitive collagen and fibronectin motif (rCF) peptide containing multiple repeats of Gly-Leu-Lys-Gly-Glu-Asn-Arg-Gly-Asp sequence derived from the cell adhesion motifs of collagen and fibronectin was produced as the alternative agent to induce cell-scaffold interaction. The DNA fragment encoding rCF peptide was amplified by a polymerase chain reaction using overlap primers without a DNA template, cloned into a protein expression vector, and expressed as a His-tag fusion peptide in Escherichia coli. The purified rCF peptide possessed cell adhesion activity about 1.5-fold of the commercial RGD peptide. The rCF peptide was grafted onto the electrospun PCL scaffold via RF plasma of Ar/O2 discharge and acrylic acid treatment. The immobilized rCF peptide significantly increased surface hydrophilicity and enhanced cell proliferation of the electrospun PCL scaffold. These findings suggest the potential application of rCF peptide for improving the biomimetic functions of polymeric scaffolds for tissue engineering.

  16. Combining a micro/nano-hierarchical scaffold with cell-printing of myoblasts induces cell alignment and differentiation favorable to skeletal muscle tissue regeneration.

    Science.gov (United States)

    Yeo, Miji; Lee, Hyeongjin; Kim, Geun Hyung

    2016-09-16

    Biomedical scaffolds must be used in tissue engineering to provide physical stability and topological/biochemical properties that directly affect tissue regeneration. In this study, a new cell-laden scaffold was developed that supplies micro/nano-topological cues and promotes efficient release of cells. The hierarchical structure consisted of poly(ε-caprolactone) macrosized struts for sustaining a three-dimensional structural shape, aligned nanofibers obtained with optimized electrospinning, and cell-printed myoblasts. Importantly, the printed myoblasts were fully safe and were efficiently released from the cell-laden struts to neighboring nanofiber networks. The incorporation of micro/nanofibers in the hierarchical scaffold significantly affected myoblast proliferation, alignment, and even facilitated the formation of myotubes. We observed that myosin heavy chain expression and the expression levels of various myogenic genes (MyoD, myogenin, and troponin T) were significantly affected by the fiber alignment achieved in our hierarchical cell-laden structure. We believe that the combination of cell-printing and a hierarchical scaffold that encourages fiber alignment is a highly promising technique for skeletal muscle tissue engineering.

  17. Preparation of three-layered porous PLA/PEG scaffold: relationship between morphology, mechanical behavior and cell permeability.

    Science.gov (United States)

    Scaffaro, R; Lopresti, F; Botta, L; Rigogliuso, S; Ghersi, G

    2016-02-01

    Interface tissue engineering (ITE) is used to repair or regenerate interface living tissue such as for instance bone and cartilage. This kind of tissues present natural different properties from a biological and mechanical point of view. With the aim to imitating the natural gradient occurring in the bone-cartilage tissue, several technologies and methods have been proposed over recent years in order to develop polymeric functionally graded scaffolds (FGS). In this study three-layered scaffolds with a pore size gradient were developed by melt mixing polylactic acid (PLA) and two water-soluble porogen agents: sodium chloride (NaCl) and polyethylene glycol (PEG). Pore dimensions were controlled by NaCl granulometry while PEG solvation created a micropores network within the devices. Scaffolds were characterized from a morphological and mechanical point of view in order to find a correlation between the preparation method, the pore architecture and compressive mechanical behavior. Biological tests were also performed in order to study the effect of pore size gradient on the permeation of different cell lines in co-culture. To imitate the physiological work condition, compressive tests were also performed in phosphate buffered saline (PBS) solution at 37°C. The presented preparation method permitted to prepare three-layered scaffolds with high control of porosity and pore size distribution. Furthermore mechanical behaviors were found to be strongly affected by pore architecture of tested devices as well as the permeation of osteoblast and fibroblast in-vitro.

  18. Scaffolds for 3D in vitro culture of neural lineage cells.

    Science.gov (United States)

    Murphy, Ashley R; Laslett, Andrew; O'Brien, Carmel M; Cameron, Neil R

    2017-03-01

    Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research.

  19. Patterned carbon nanotubes as a new three-dimensional scaffold for mesenchymal stem cells.

    Science.gov (United States)

    Bitirim, Verda Ceylan; Kucukayan-Dogu, Gokce; Bengu, Erman; Akcali, Kamil Can

    2013-07-01

    We investigated the cellular adhesive features of mesenchymal stem cells (MSC) on non-coated and collagen coated patterned and vertically aligned carbon nanotube (CNT) structures mimicking the natural extra cellular matrix (ECM). Patterning was achieved using the elasto-capillary induced by water treatment on the CNT arrays. After confirmation with specific markers both at transcript and protein levels, MSCs from different passages were seeded on either collagen coated or non-coated patterned CNTs. Adhesion and growth of MSCs on the patterned CNT arrays were examined using scanning electron microscopy image analysis and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. The highest MSC count was observed on the non-coated patterned CNTs at passage zero, while decreasing numbers of MSCs were found at the later passages. Similarly, MTT assay results also revealed a decrease in the viability of the MSCs for the later passages. Overall, the cell count and viability experiments indicated that MSCs were able to better attach to non-coated patterned CNTs compared to those coated with collagen. Therefore, the patterned CNT surfaces can be potentially used as a scaffold mimicking the ECM environment for MSC growth which presents an alternative approach to MSC-based transplantation therapy applications.

  20. Protocol and cell responses in three-dimensional conductive collagen gel scaffolds with conductive polymer nanofibres for tissue regeneration.

    Science.gov (United States)

    Sirivisoot, Sirinrath; Pareta, Rajesh; Harrison, Benjamin S

    2014-02-06

    It has been established that nerves and skeletal muscles respond and communicate via electrical signals. In regenerative medicine, there is current emphasis on using conductive nanomaterials to enhance electrical conduction through tissue-engineered scaffolds to increase cell differentiation and tissue regeneration. We investigated the role of chemically synthesized polyaniline (PANI) and poly(3,4-ethylenedioxythiophene) (PEDOT) conductive polymer nanofibres for conductive gels. To mimic a naturally derived extracellular matrix for cell growth, type I collagen gels were reconstituted with conductive polymer nanofibres and cells. Cell viability and proliferation of PC-12 cells and human skeletal muscle cells on these three-dimensional conductive collagen gels were evaluated in vitro. PANI and PEDOT nanofibres were found to be cytocompatible with both cell types and the best results (i.e. cell growth and gel electrical conductivity) were obtained with a low concentration (0.5 wt%) of PANI. After 7 days of culture in the conductive gels, the densities of both cell types were similar and comparable to collagen positive controls. Moreover, PC-12 cells were found to differentiate in the conductive hydrogels without the addition of nerve growth factor or electrical stimulation better than collagen control. Importantly, electrical conductivity of the three-dimensional gel scaffolds increased by more than 400% compared with control. The increased conductivity and injectability of the cell-laden collagen gels to injury sites in order to create an electrically conductive extracellular matrix makes these biomaterials very conducive for the regeneration of tissues.

  1. Human dental pulp stem cells and gingival fibroblasts seeded into silk fibroin scaffolds have the same ability in attracting vessels

    Directory of Open Access Journals (Sweden)

    Anna eWoloszyk

    2016-04-01

    Full Text Available Neovascularization is one of the most important processes during tissue repair and regeneration. Current healing approaches based on the use of biomaterials combined with stem cells in critical-size bone defects fail due to the insufficient implant vascularization and integration into the host tissues. Therefore, here we studied the attraction, ingrowth, and distribution of blood vessels from the chicken embryo chorioallantoic membrane into implanted silk fibroin scaffolds seeded with either human dental pulp stem cells or human gingival fibroblasts. Perfusion capacity was evaluated by non-invasive in vivo Magnetic Resonance Imaging while the number and density of blood vessels were measured by histomorphometry. Our results demonstrate that human dental pulp stem cells and gingival fibroblasts possess equal abilities in attracting vessels within silk fibroin scaffolds. Additionally, the prolonged in vitro pre-incubation period of these two cell populations favors the homogeneous distribution of vessels within silk fibroin scaffolds, which further improves implant survival and guarantees successful healing and regeneration.

  2. Compatibility of Porous Chitosan Scaffold with the Attachment and Proliferation of human Adipose-Derived Stem Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Gomathysankar S

    2016-11-01

    Full Text Available Adipose-derived stem cells (ASCs have potential applications in the repair and regeneration of various tissues and organs. The use of various scaffold materials as an excellent template for mimicking the extracellular matrix to induce the attachment and proliferation of different cell types has always been of interest in the field of tissue engineering because ideal biomaterials are in great demand. Chitosan, a marine polysaccharide, have wide clinical applications and it acts as a promising scaffold for cell migration and proliferation. ASCs, with their multi-differentiation potential, and chitosan, with its great biocompatibility with ASCs, were investigated in the present study. ASCs were isolated and were characterized by two different methods: immunocytochemistry and flow cytometry, using the mesenchymal stem cell markers CD90, CD105, CD73 and CD29. The ASCs were then induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. These ASCs were incorporated into a porous chitosan scaffold (PCS, and their structural morphology was studied using a scanning electron microscope and hematoxylin and eosin staining. The proliferation rate of the ASCs on the PCS was assessed using a PrestoBlue viability assay. The results indicated that the PCS provides an excellent template for the adhesion and proliferation of ASCs. Thus, this study revealed that PCS is a promising biomaterial for inducing the proliferation of ASCs, which could lead to successful tissue reconstruction in the field of tissue engineering.

  3. Cell-seeded scaffolds based on poly(ethylene oxide) and poly(butylene terephthalate) block copolymers for bone tissue engineering

    OpenAIRE

    Claase, Menno Bernard

    2004-01-01

    This thesis describes the development of polymeric scaffolds containing bone marrow stromal cells (BMSCs) that are cultured in an osteogenic medium and can be used for the formation of functional bone tissue upon implantation

  4. Basic fibroblast growth factor-loaded, mineralized biopolymer-nanofiber scaffold improves adhesion and proliferation of rat mesenchymal stem cells.

    Science.gov (United States)

    Kim, Tae-Hyun; Kim, Jung-Ju; Kim, Hae-Won

    2014-02-01

    Nanofibrous matrices are attractive scaffolding platforms for tissue regeneration. Modification of the nanofiber surface, particularly with biological proteins, improves cellular interactions. Here, we loaded basic fibroblast growth factor (bFGF) onto mineralized nanofibers and investigated the effect on adhesion and proliferation of rat mesenchymal stem cells. bFGF loading was significantly higher on the mineralized nanofiber than on the non-mineralized one. Release of bFGF from the mineralized nanofibers was continuous over 2 weeks. Cells cultured on the bFGF-loaded nanofiber attached and proliferated in significantly higher numbers than those on the bFGF-free nanofiber. bFGF-receptor inhibition study confirmed the biological role played by the loaded bFGF. This study details the advantages of the mineralized nanofiber surface for the loading and delivery bFGF, and thus the bFGF-loaded nanofiber scaffold may be useful for tissue repair and regeneration.

  5. Potential of centrifugal seeding method in improving cells distribution and proliferation on demineralized cancellous bone scaffolds for tissue-engineered meniscus.

    Science.gov (United States)

    Zhang, Zheng-Zheng; Jiang, Dong; Wang, Shao-Jie; Qi, Yan-Song; Zhang, Ji-Ying; Yu, Jia-Kuo

    2015-07-22

    Tissue-engineered meniscus offers a possible solution to the regeneration and replacement problem of meniscectomy. However, the nonuniform distribution and declined proliferation of seeded cells on scaffolds hinder the application of tissue-engineered meniscus as a new generation of meniscus graft. This study systematically investigated the performances of different seeding techniques by using the demineralized cancellous bone (DCB) as the scaffold. Static seeding, injection seeding, centrifugal seeding, and vacuum seeding methods were used to seed the meniscal fibrochondrocytes (MFCs) and mesenchymal stem cells (MSCs) to scaffolds. Cell-binding efficiency, survival rate, distribution ability, and long-term proliferation effects on scaffolds were quantitatively evaluated. Cell adhesion was compared via cell-binding kinetics. Cell viability and morphology were assessed by using fluorescence staining. Combined with the reconstructed three-dimensional image, the distribution of seeded cells was investigated. The Cell Counting Kit-8 assay and DNA assay were employed to assess cell proliferation. Cell-binding kinetics and cell survival of the MFCs were improved via centrifugal seeding compared to injection or vacuum seeding methods. Seeded MFCs by centrifugation showed a more homogeneous distribution throughout the scaffold than cells seeded by other methods. Moreover, the penetration depth in the scaffold of seeded MFCs by centrifugation was 300-500 μm, much higher than the value of 100-300 μm by the surface static and injection seeding. The long-term proliferation of the MFCs in the centrifugal group was also significantly higher than that in the other groups. The results of the MSCs were similar to those of the MFCs. The centrifugal seeding method could significantly improve MFCs or MSCs distribution and proliferation on the DCB scaffolds, thus providing a simple, cost-effective, and effective cell-seeding protocol for tissue-engineered meniscus.

  6. Addition of selenium nanoparticles to electrospun silk scaffolds improves mammalian cell activity while reducing bacterial growth

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    Stanley Chung

    2016-07-01

    Full Text Available Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas compared to non-electrospun equivalents. However, purified silk promotes microbial growth. In contrast, selenium nanoparticles have excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics.

  7. Experimental study on cultivation and purification of bone marrow-derived mesenchymal stem cells and its co-culture with chitosan porous scaffolds in vitro

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    Feng YAN

    2014-12-01

    Full Text Available Background As commonly used scaffold material in tissue engineering, chitosan has many advantages, such as strong biodegradability, low antigenicity, good biocompatibility and no pyrogen reaction. This study aims to isolate, cultivate and purify Sprague-Dawley (SD rat bone marrow-derived mesenchymal stem cells (BMSCs, and to observe the growth of BMSCs when co-cultured with self-made chitosan porous scaffold in vitro and to test the biocompatibility of this tissue engineering scaffold, so as to lay the foundation for promoting nerve regeneration of transplant treatment.  Methods Three-week-old healthy male SD rats were used in this study, and BMSCs were isolated and purified through bone marrow adherent culture method. The surface markers of BMSCs at Passage 3 were detected and identified by flow cytometry (FCM and the BMSCs were three?dimensionally cultured in vitro on chitosan porous scaffolds produced by freeze-drying method. Ethanol alternative method was used to detect the chitosan scaffold porosity. Scanning electron microscope was used to explore the internal structure of the scaffold, measure the size of its aperture, and observe the morphology and development of the cells within the scaffold. Methyl thiazolyl tetrazolium (MTT method was used to determine the cells' proliferation.  Results The cultured BMSCs were uniform and similiar to fibrous arrangement, and mixed cells reduced obviously. The identification result of FCM showed the CD29 positive rate was 98.49% and CD45RA positive rate was only 0.85%. The chitosan scaffold had an interlinked, uniform similar three-dimensional porous structure and its aperture porosity was 90%. Some cells stretched out pseudopod and infiltrated into the porous structure of scaffold, even fusing with them. The BMSCs were seeded in the scaffold successfully. The chitosan scaffold had no obvious effect on BMSCs' proliferation. Conclusions Chitosan porous scaffolds have good structural character and

  8. Porous Lactose-Modified Chitosan Scaffold for Liver Tissue Engineering: Influence of Galactose Moieties on Cell Attachment and Mechanical Stability

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    Birong Wang

    2016-01-01

    Full Text Available Galactosylated chitosan (CTS has been widely applied in liver tissue engineering as scaffold. However, the influence of degree of substitution (DS of galactose moieties on cell attachment and mechanical stability is not clear. In this study, we synthesized the lactose-modified chitosan (Lact-CTS with various DS of galactose moieties by Schiff base reaction and reducing action of NaBH4, characterized by FTIR. The DS of Lact-CTS-1, Lact-CTS-2, and Lact-CTS-3 was 19.66%, 48.62%, and 66.21% through the method of potentiometric titration. The cell attachment of hepatocytes on the CTS and Lact-CTS films was enhanced accompanied with the increase of galactose moieties on CTS chain because of the galactose ligand-receptor recognition; however, the mechanical stability of Lact-CTS-3 was reduced contributing to the extravagant hydrophilicity, which was proved using the sessile drop method. Then, the three-dimensional Lact-CTS scaffolds were fabricated by freezing-drying technique. The SEM images revealed the homogeneous pore bearing the favorable connectivity and the pore sizes of scaffolds with majority of 100 μm; however, the extract solution of Lact-CTS-3 scaffold significantly damaged red blood cells by hemolysis assay, indicating that exorbitant DS of Lact-CTS-3 decreased the mechanical stability and increased the toxicity. To sum up, the Lact-CTS-2 with 48.62% of galactose moieties could facilitate the cell attachment and possess great biocompatibility and mechanical stability, indicating that Lact-CTS-2 was a promising material for liver tissue engineering.

  9. Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

    OpenAIRE

    Costa, Pedro Ferreira da

    2014-01-01

    Programa Doutoral em Engenharia Biomédica In a typical tissue engineering approach, cells are collected from the patient and then seeded into a threedimensional scaffold where they proliferate to generate a tissue-like substitute to be re-implanted back into the defect site. However, human tissues possess various degrees of complexity which often makes them impossible to be reproduced in such a simplified way. In fact, many tissues such as bone, for example, exhibit specific ar...

  10. Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via Recruiting Endogenous Stem Cells.

    Science.gov (United States)

    Shi, Jiajia; Sun, Jie; Zhang, Wen; Liang, Hui; Shi, Qin; Li, Xiaoran; Chen, Yanyan; Zhuang, Yan; Dai, Jianwu

    2016-10-07

    The reconstruction of bone usually depends on substitute transplantation, which has drawbacks including the limited bone substitutes available, comorbidity, immune rejection, and limited endogenous bone regeneration. Here, we constructed a functionalized bone substitute by combining application of the demineralized bone matrix (DBM) and collagen-binding stromal-cell-derived factor-1α (CBD-SDF-1α). DBM was a poriferous and biodegradable bone substitute, derived from bovine bone and consisting mainly of collagen. CBD-SDF-1α could bind to collagen and be controllably released from the DBM to mobilize stem cells. In a rat femur defect model, CBD-SDF-1α-modified DBM scaffolds could efficiently mobilize CD34(+) and c-kit(+) endogenous stem cells homing to the injured site at 3 days after implantation. According to the data from micro-CT, CBD-SDF-1α-modified DBM scaffolds could help the bone defects rejoin with mineralization accumulated and bone volume expanded. Interestingly, osteoprotegerin (OPG) and osteopontin (OPN) were highly expressed in CBD-SDF-1α group at an early time after implantation, while osteocalcin (OCN) was more expanded. H&E and Masson's trichrome staining showed that the CBD-SDF-1α-modified DBM scaffold group had more osteoblasts and that the bone defect rejoined earlier. The ultimate strength of the regenerated bone was investigated by three-point bending, showing that the CBD-SDF-1α group had superior strength. In conclusion, CBD-SDF-1α-modified DBM scaffolds could promote bone regeneration by recruiting endogenous stem cells.

  11. IQGAP1: A microtubule-microfilament scaffolding protein with multiple roles in nerve cell development and synaptic plasticity.

    Science.gov (United States)

    Jausoro, Ignacio; Mestres, Iván; Remedi, Mónica; Sanchez, Mónica; Cáceres, Alfredo

    2012-11-01

    In this article, we review our current understanding of the biology of IQ domain-containing GTPase-Activating Protein 1, IQGAP1, a scaffolding protein with multiple binding partners, which is widely expressed among different cell types, including neurons, and capable of linking Rho-GTPase signaling with cytosleletal elements and environmental cues. Interestingly, a series of recent studies suggest that IQGAP family members have an important role in neuronal development, synaptic plasticity and nervous system disorders involving alterations in spine density.

  12. Regeneration of meniscus tissue using adipose mesenchymal stem cells-chondrocytes co-culture on a hybrid scaffold: In vivo study.

    Science.gov (United States)

    Moradi, Lida; Vasei, Mohammad; Dehghan, Mohammad M; Majidi, Mohammad; Farzad Mohajeri, Saeed; Bonakdar, Shahin

    2017-05-01

    The meniscus has poor intrinsic regenerative capacity and its damage inevitably leads to articular cartilage degeneration. We focused on evaluating the effects of Polyvinyl alcohol/Chitosan (PVA/Ch) scaffold seeded by adipose-derived mesenchymal stem cell (ASC) and articular chondrocytes (AC) in meniscus regeneration. The PVA/Ch scaffolds with different molar contents of Ch (Ch1, Ch2, Ch4 and Ch8) were cross-linked by pre-polyurethane chains. By increasing amount of Ch tensile modulus was increased from 83.51 MPa for Ch1 to 110 MPa for Ch8 while toughness showed decrease from 0.33 mJ/mm(3) in Ch1 to 0.11 mJ/mm(3) in Ch8 constructs. Moreover, swelling ratio and degradation rate increased with an increase in Ch amount. Scanning electron microscopy imaging was performed for pore size measurement and cell attachment. At day 21, Ch4 construct seeded by AC showed the highest expression with 24.3 and 22.64 folds increase in collagen II and aggrecan (p ≤ 0.05), respectively. Since, the mechanical properties, water uptake and degradation rate of Ch4 and Ch8 compositions had no statistically significant differences, Ch4 was selected for in vivo study. New Zealand rabbits were underwent unilateral total medial meniscectomy and AC/scaffold, ASC/scaffold, AC-ASC (co-culture)/scaffold and cell-free scaffold were engrafted. At 7 months post-implantation, macroscopic, histologic, and immunofluorescent studies for regenerated meniscus revealed better results in AC/scaffold group followed by AC-ASC/scaffold and ASC/scaffold groups. In the cell-free scaffold group, there was no obvious meniscus regeneration. Articular cartilages were best preserved in AC/scaffold group. The best histological score was observed in AC/scaffold group. Our results support that Ch4 scaffold seeded by AC alone can successfully regenerate meniscus in tearing injury and ASC has no significant contribution in the healing process.

  13. Preliminary In Vitro Assessment of Stem Cell Compatibility with Cross-Linked Poly(ε-caprolactone urethane Scaffolds Designed through High Internal Phase Emulsions

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    Sylvie Changotade

    2015-01-01

    Full Text Available By using a high internal phase emulsion process, elastomeric poly(ε-caprolactone urethane (PCLU scaffolds were designed with pores size ranging from below 150 μm to 1800 μm and a porosity of 86% making them suitable for bone tissue engineering applications. Moreover, the pores appeared to be excellently interconnected, promoting cellularization and future bone ingrowth. This study evaluated the in vitro cytotoxicity of the PCLU scaffolds towards human mesenchymal stem cells (hMSCs through the evaluation of cell viability and metabolic activity during extract test and indirect contact test at the beginning of the scaffold lifetime. Both tests demonstrated that PCLU scaffolds did not induce any cytotoxic response. Finally, direct interaction of hMSCs and PCLU scaffolds showed that PCLU scaffolds were suitable for supporting the hMSCs adhesion and that the cells were well spread over the pore walls. We conclude that PCLU scaffolds may be a good candidate for bone tissue regeneration applications using hMSCs.

  14. Differentiation capacity and maintenance of differentiated phenotypes of human mesenchymal stromal cells cultured on two distinct types of 3D polymeric scaffolds.

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    Leferink, A M; Santos, D; Karperien, M; Truckenmüller, R K; van Blitterswijk, C A; Moroni, L

    2015-12-01

    Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.

  15. Electroactive Tissue Scaffolds with Aligned Pores as Instructive Platforms for Biomimetic Tissue Engineering