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ABSTRACT: Chitosan-gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan-gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5months. The amount of glycosaminoglycans in C1G1WSC constructs (0.187±0.095μg/mg tissue) harvested from the animals after 6.5months was 14wt.% of that in normal cartilage (1.329±0.660μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5months was about 0.539MPa, which approached to that of normal cartilage (0.735MPa), while that in the blank control (3.881MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.
Materials science & engineering. C, Materials for biological applications. 07/2013; 33(5):2855-63.
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ABSTRACT: Bacterial cellulose (BC) and bacterial cellulose-chitosan (BC-Ch) membranes were successfully produced in large scale. BC was synthesized by Acetobacter xylinum. BC-Ch was prepared by immersing BC in chitosan followed by freeze-drying. The surface morphology of BC and BC-Ch membranes were examined by a scanning electron microscope (SEM). SEM images showed that BC-Ch possessed a denser fibril network with smaller pores than BC. Infrared spectroscopy was used to confirm the incorporation of chitosan in BC-Ch. The swelling behavior, water retention capacity, and mechanical properties of BC and BC-Ch were further evaluated. Results indicated that both membranes maintained proper moisture contents for an extensive period without dehydration. The tensile strength and elongation at break for BC-Ch were slightly lower while the Young's modulus was higher. Cell culture studies demonstrated that BC and BC-Ch had no cytotoxicity. In the antibacterial test, the addition of chitosan in BC showed significant growth inhibition against Escherichia coli and Staphylococcus aureus. The effects of BC and BC-Ch on skin wound healing were assessed by rat models. Histological examinations revealed that wounds treated with BC-Ch epithelialized and regenerated faster than those treated with BC or Tegaderm. Therefore, BC-Ch was considered as a potential candidate for wound dressing materials.
Carbohydrate polymers. 04/2013; 94(1):603-11.
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ABSTRACT: A unique biomaterial-based system was developed to generate dynamic three-dimensional (3D) multicellular spheroids of mesenchymal stem cells (MSCs). MSCs were cultured on transparent membranes made of chitosan or those further grafted with hyaluronan (HA) in different densities. MSCs vigorously migrated and were self-assembled into highly mobile 3D spheroids with substrate-dependent upregulation of adhesion molecule N-cadherin. MSC spheroids showed increased expression of Wnt genes/proteins and substrate-dependent cell fate. The correlation of differentiation capacities with Wnt signaling and crosstalk with other pathways such as ERK1/2 or Smad2/3 were observed for MSC spheroids but not for the conventional 2D cultured cells. Wnt3a-mediated canonical Wnt signaling was more active for MSC spheroids derived on chitosan, which were prone to osteogenesis. Wnt5a-mediated non-canonical Wnt signaling was more active for MSC spheroids derived on HA-grafted chitosan, which were prone to chondrogenesis. In particular, the relative importance of Wnt5a-mediated non-canonical vs. Wnt3a-mediated canonical Wnt signals in determining the cell fate was controlled by the grafting density of HA on chitosan. Treatment with the inhibitor of canonical Wnt-associated signaling molecules suppressed the osteogenesis of MSC spheroids on chitosan. This study demonstrates that Wnt signaling of MSCs is distinct in 3D environment and is substrate-dependent. The convenient 3D platform may be used to examine the role of Wnt signaling in controlling MSC fate under different extracellular environments, and potentially applied to study stem cell behavior in regenerative medicine, normal development, and cancer.
Biomaterials 04/2013; · 7.40 Impact Factor
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ACS Applied Materials & Interfaces 03/2013; · 4.53 Impact Factor
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ABSTRACT: Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp-containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell-based therapy in myocardial infarction.
BioResearch open access. 02/2013; 2(1):28-39.
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ABSTRACT: Silver nanoparticles (AgNPs) are known for their bactericidal abilities. The antibacterial potency is dependent on the particle size and dispersion status. In this study, we synthesized AgNP/NSP nanohybrids in two different weight ratios (1/99 and 8/92) using the fully exfoliated clay, i.e. nanosilicate platelets (NSP), as a dispersing agent and carrier for AgNPs. Due to the size of NSP, the immobilized AgNPs do not enter cells readily, which may lower the risk associated with the cellular uptake of AgNPs. The biocompatibility, immunological response, and antimicrobial activities of AgNP/NSP hybrids were evaluated. The results revealed that AgNP/NSP hybrids elicited merely mild inflammatory response and retained the outstanding antibacterial activity. The hybrids were further embedded in poly(ether)urethane (PEU) to increase the biocompatibility. At the same silver content (20 ppm), the PEU-AgNP/NSP nanocomposites were nontoxic to mouse skin fibroblasts, while simultaneously exhibited nearly complete bacterial growth reduction (99.9%). PEU containing the same amount of free AgNPs did not display such an effect. Our results verify the better biosafety of the AgNPs/NSP hybrids and their polymer nanocomposites for further clinical use.
ACS Applied Materials & Interfaces 12/2012; · 4.53 Impact Factor
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ABSTRACT: Cell transplantation is a useful therapy for treating peripheral nerve injuries. The clinical use of Schwann cells (SCs), however, is limited because of their limited availability. An emerging solution to promote nerve regeneration is to apply injured nerves with stem cells derived from various tissues. In this study, different types of allogenic cells including SCs, adipose-derived adult stem cells (ASCs), dental pulp stem cells (DPSCs) and the combination of SCs with ASCs or DPSCs were seeded on nerve conduits to test their efficacy in repairing a 15mm long critical gap defect of rat sciatic nerve. The regeneration capacity and functional recovery were evaluated by the histological staining, electrophysiology, walking track and functional gait analysis after 8 weeks of implantation. An in vitro study was also performed to verify if the combination of cells led to synergistic neurotrophic effects (NGF, BDNF and GDNF). Experimental rats receiving conduits seeded with a combination of SCs and ASCs had the greatest functional recovery, as evaluated by the walking track, functional gait, nerve conduction velocity (NCV) and histological analysis. Conduits seeded with cells were always superior to the blank conduits without cells. Regarding NCV and the number of blood vessels, conduits seeded with SCs and DPSCs exhibited better values than those seeded with DPSCs only. Results from the in vitro study confirmed the synergistic NGF production from the coculture of SCs and ASCs. It was concluded that co-culture of SCs with ASCs or DPSCs in conduit promoted peripheral nerve regeneration over a critical gap defect.
Cell Transplantation 11/2012; · 5.13 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) were recently found to form three-dimensional (3D) multicellular spheroids on chitosan membranes. The exact mechanism of spheroid formation, however, remains unclear. In this study, the regulation of spheroid formation for adipose derived adult stem cells (ADAS) grown on chitosan membranes was examined. By varying the membrane thickness, calcium concentration in culture medium, and acetylation extent of chitosan, the physico-chemical characteristics of chitosan that modulated spheroid formation was elucidated. The capacity of cardiomyogenic differentiation was further evaluated. Results suggested that the calcium binding capacity of chitosan may affect the cell-substrate and cell-cell interactions and critically influence the dynamics of spheroid formation. The intracellular calcium level was elevated for ADAS spheroids on chitosan. Chitosan-bound calcium was observed to enter the cells. The expression of N-cadherin was upregulated for ADAS spheroids on chitosan, evidenced by quantitative RT-PCR and Western blot. After the induction by 5-aza, the expression levels of cardiac marker genes (Gata4, Nkx2.5, Tnnt2, and Myh6) were remarkably enhanced (about four-fold) for ADAS on chitosan vs. tissue culture polystyrene or polyvinyl alcohol. Immunofluorescence staining confirmed the expression of cardiac-associated tight junction protein ZO-1 for ADAS grown on chitosan membranes. The gene expression of Wnt11 was significantly upregulated for ADAS spheroids on chitosan at 3 days and 12 days. We suggested that Wnt11 may be involved in the spheroid formation and cardiomyogenic differentiation of MSCs on chitosan membranes. Spheroids formed on the acetylated chitosan or polyvinyl alcohol membranes failed to show such behavior. The properties of MSC spheroids were therefore determined by the culture substrate.
Biomaterials 09/2012; 33(35):8943-54. · 7.40 Impact Factor
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ABSTRACT: Nanoparticles (NPs) are usually surface modified to increase endocytosis for applications in cellular imaging and gene delivery. The influence of cell culture substrates on endocytosis remains relatively unexplored. This study investigated the substrate-mediated effects on the uptake of NPs by mesenchymal stem cells (MSCs). Two types of NPs were employed, negatively charged paramagnetic iron oxide (Fe(3)O(4)) NPs (~5 nm) and bare plasmid DNA pTRE-Tight-DsRED2 (3.3 kb, ~5 nm), each of which were poorly endocytosed by the adipose-derived MSCs grown on tissue culture polystyrene (TCPS). When cells were cultured on chitosan or hyaluronan-modified chitosan (chitosan-HA) membranes, significant increases (>5-fold) in the intracellular uptake of Fe(3)O(4) NPs as well as transfectability of plasmid DNA were demonstrated. The enhancement in transgene expression was more pronounced than that using the transfection agent. The beneficial effects were not caused by elevated proliferation or a change in the differentiation state of interacting MSCs. On chitosan and chitosan-HA, cells moved fast and formed spheroids. The cytoskeletal arrangement associated with the up-regulated RhoA activity during spheroid formation may have accounted for the increased endocytosis. Using different inhibitors, the endocytosis pathways were further clarified. Both Fe(3)O(4) NPs and plasmid DNA were taken up primarily by clathrin-mediated endocytosis on chitosan (~50%). The caveolae-mediated endocytosis on chitosan-HA was more evident (~30-40%) than that on chitosan (<25%). For plasmid DNA but not Fe(3)O(4) NPs, macropinocytosis also occurred on both substrates. Chitosan and chitosan-HA as cell culture substrates may activate different endocytic pathways of MSCs to increase NP internalization or plasmid transfection. The substrate-mediated endocytosis described here may represent a new and potentially attractive approach to facilitate stem cell labeling or to improve gene delivery efficiency without altering cell viability and differentiation.
Biomaterials 05/2012; 33(14):3639-50. · 7.40 Impact Factor
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ABSTRACT: Silver nanoparticles (AgNPs) are known for their excellent antibacterial activities. The possible toxicity, however, is a major concern for their applications. Three types of AgNPs were prepared in this study by chemical processes. Each was stabilized by a polymer surfactant, which was expected to reduce the exposure of cells to AgNPs and therefore their cytotoxicity. The polymer stabilizers included poly(oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol) (PVA). The cytotoxicity of these chemically produced AgNPs to mouse skin fibroblasts (L929), human hepatocarcinoma cells (HepG2), and mouse monocyte macrophages (J774A1) was compared to that of physically produced AgNPs and gold nanoparticles (AuNPs) as well as the standard reference material RM8011 AuNPs. Results showed that SMA-AgNPs were the least cytotoxic among all materials, but cytotoxicity was still observed at higher silver concentrations (>30 ppm). Macrophages demonstrated the inflammatory response with cell size increase and viability decrease upon exposure to 10 ppm of the chemically produced AgNPs. SMA-AgNPs did not induce hemolysis at a silver concentration below 1.5 ppm. Regarding the antibacterial activity, POEM-AgNPs and SMA-AgNPs at 1 ppm silver content showed 99.9% and 99.3% growth inhibition against E. coli, while PVA-AgNPs at the same silver concentration displayed 79.1% inhibition. Overall, SMA-AgNPs demonstrated better safety in vitro and greater antibacterial effects than POEM-AgNPs and PVA-AgNPs. This study suggested that polymer stabilizers may play an important role in determining the toxicity of AgNPs.
Nanotechnology 02/2012; 23(6):065102. · 3.98 Impact Factor
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ABSTRACT: Chitosan is a nontoxic, biodegradable and biocompatible polymer. Rapid prototyped chitosan scaffolds were manufactured by liquid-frozen deposition of chitosan fibers in this study. To investigate if the air plasma (AP) treatment could be used to improve the surface properties of these scaffolds for cell attachment, chitosan films were first prepared and treated with AP under different conditions. Under the optimized condition, the water contact angle of chitosan films was significantly reduced from 90 ± 1° to 19 ± 1° after AP treatment. On the other hand, the surface charge and nanometric roughness of chitosan films increased after AP treatment. X-ray photoelectron spectroscopy measurement on AP-treated three-dimensional chitosan scaffolds showed that nitrogen and oxygen increased at each location inside the scaffolds as compared to the untreated ones, which indicated that AP could permeate through the fibrous stacks of the scaffolds and effectively modify the interior (visible) surface of the scaffolds. Moreover, AP treatment enabled the migration of MC3T3-E1 cells into the scaffolds, facilitated their proliferation and promoted the bone mineral deposition. These results suggested that fibers-stacked chitosan scaffolds may be produced by liquid-frozen deposition and treated with AP for bone tissue engineering applications.
Biofabrication 01/2012; 4(1):015002. · 3.48 Impact Factor
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ABSTRACT: Literature has different opinions regarding the percentage of mesenchymal stem cell (MSC)-like population in human gingival tissue. Isolation of these cells is thus important for clinical applications. In this study, two typical but distinct types of gingival fibroblasts (GF), GF-A and GF-B, were grown from human gingival biopsies. They were characterized for surface markers by flow cytometry as well as the expressions of stemness and neural crest marker genes by RT-PCR. The two types of GF were slightly different in their surface markers; however, they had dramatic difference in the expression levels of stemness marker genes and neural crest marker genes. They also demonstrated distinct differentiation capacity. Upon the appropriate induction, GF-A were capable of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation while GF-B only underwent osteogenic differentiation. By culturing either type of GF on chitosan membranes for 24 h, we were able to isolate two distinct subpopulations in each type of GF, i.e. cells with spheroid-forming ability (GF-AS and GF-BS) or those remained flat and attached (GF-AN and GF-BN). We further characterized these cells, and determined the common properties shared by the spheroid-forming subpopulation "S", as well as those shared by the non-spheroid-forming subpopulation "N". The subpopulation "S" was capable of the multilineage differentiation, while the subpopulation "N" was only efficient in osteogenic differentiation. GF-A and GF-B had different proportions of subpopulations. Chitosan as the cell culture substratum up-regulated the N-cadherin expression of the "S" but not "N" subpopulation, which may account for the cell sorting effect. This study showed that chitosan membranes could be used for isolation of the spheroid forming subpopulation in human GF that contained multipotent adult stem cells of which the number varied among donors and sites.
Biomaterials 01/2012; 33(9):2642-55. · 7.40 Impact Factor
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ABSTRACT: Model surfaces of polyurethane-gold nanocomposites (PU-Au) were used to examine cell behavior on nanophase-segregated materials. Previously we showed that endothelial cell (EC) migration on these materials was modulated by the PI3K/Akt/eNOS pathway. The present study, investigated the expressions of alpha5/beta3 (α5β3) integrin, focal adhesion kinase (FAK), and other downstream signal molecules such as the Rho family and matrix metalloproteinases 2 (MMP-2) induced by the materials in two different cells, that is bovine arterial endothelial cells (BAEC) and human skin fibroblasts (HSF). Both cells proliferated better on the more phase-separated PU-Au 43.5 ppm than on the less phase-separated controls (PU and PU-Au 174 ppm). On PU-Au 43.5 ppm, BAEC compared to HSF had denser actin fibers and were more extended. BAEC became rounded with Y-27632 treatment and shrunk with LY294002 treatment. Treatment by inhibitors only caused slight changes in HSF. The migration distance of BAEC on PU-Au 43.5 ppm was greater than that of HSF, and was significantly reduced by LY294002 or Y-27632 but not SU-1498. The expressions of p-FAK, p-RhoA, p-Rac/Cdc42, MMP2, and α5β3 integrin induced by PU-Au 43.5 ppm were more pronounced in BAEC versus HSF. Further enhancement in MMP2 and α5β3 integrin expressions by FAK-GFP transfection was more remarkable for cells on PU-Au 43.5 ppm. Our findings suggested that the integrin α5β3/FAK pathway may be induced by nanophase-separated materials in both ECs and fibroblasts to promote their proliferation/migration, while the crosstalk between the PI3K/Akt/eNOS pathway and FAK/Rho-GTPase activation may account for the greater effect in ECs than in fibroblasts.
Journal of Biomedical Materials Research Part A 01/2012; 100(1):26-37. · 2.63 Impact Factor
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Shu-Her Chiao,
Siou-Hong Lin,
Ching-I Shen,
Jiunn-Wang Liao,
I-Jiuan Bau,
Jiun-Chiou Wei,
Li-Ping Tseng, Shan-hui Hsu,
Ping-Shan Lai,
Shinn-Zong Lin,
Jiang-Jen Lin,
Hong-Lin Su
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ABSTRACT: Developing effective and safe drugs is imperative for replacing antibiotics and controlling multidrug-resistant microbes. Nanoscale silicate platelet (NSP) and its nanohybrid, silver nanoparticle/NSP (AgNP/NSP), have been developed, and the nanohybrids show a strong and general antibacterial activity in vitro. Here, their efficacy for protecting Salmonella-infected chicks from fatality and septicemia was evaluated. Both orally administrated NSP and AgNP/NSP, but not AgNPs alone, effectively reduced the systemic Salmonella infection and mortality. In addition, quantitative Ag analyses demonstrated that Ag deposition from AgNP/NSP in the intestines was less than that from conventional AgNPs, indicating that the presence of NSP for immobilizing AgNPs reduced Ag accumulation in tissue and improved the safety of AgNPs. These in vivo results illustrated that both NSP and AgNP/NSP nanohybrid represent potential agents for controlling enteric bacterial infections.
International Journal of Nanomedicine 01/2012; 7:2421-32. · 3.13 Impact Factor
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ABSTRACT: A novel method to exfoliate the montmorillonite clay was developed previously to generate random nanosilicate platelets (NSP), one kind of delaminated clay. To improve their dispersion in a polymer, we modified NSPs by three types of surfactants (cationic Qa, nonionic Qb, and anionic Qc) in this study and used them to prepare nanocomposites with polyurethane (PU). The zeta potential, antimicrobial ability, and biocompatibility of these surfactant-modified NSPs (abbreviated "NSQ") were characterized. It was found that the zeta potential of Qa-modified NSP (NSQa) was positive, whereas those of NSP and the other two NSQs (NSQb and NSQc) were negative. All NSQ presented less cytotoxicity than NSP. NSQa and NSQc showed excellent antimicrobial activities against S. aureus (Gram-positive strain) and E. coli (Gram-negative strain). The nanocomposites of NSQ with PU were then characterized for surface and mechanical properties, cell attachment and proliferation, antimicrobial activity in vitro, and biocompatibility in vivo. A higher surfactant to NSP ratio was found to improve the dispersion of NSQ in PU matrix. The mechanical properties of all PU/NSQ nanocomposites were significantly enhanced. Among various NSQ, only NSQa were observed to migrate to the composite surface. The attachment and proliferation of endothelial cells and fibroblasts in vitro as well as biocompatibility in vivo were significantly better for PU/NSQa containing 1% of NSQa than other materials. The microbiostasis ratios of PU/NSQ nanocomposites containing 1% NSQa or NSQc were >90%. These results proposed the safety and potential antimicrobial applications of surfactant-modified delaminated clays and their nanocomposites with PU polymer.
ACS Applied Materials & Interfaces 11/2011; 4(1):338-50. · 4.53 Impact Factor
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ABSTRACT: Nanocomposites from a polyether-type waterborne polyurethane (PU) and 0.1 wt % of silicate materials were prepared. The individual silicate materials were natural clays (montmorillonite and mica), their exfoliated clays [nano silicate platelets (NSP) and nano mica platelets], and NSP modified with C18 fatty amine (NSP-S). The physico-chemcical properties and antimicrobial activity of the nanocomposites were characterized in vitro. The biostability and biocompatibility of the nanocomposites were evaluated in vivo. The nanocomposites exhibited various surface morphologies with phase separation of hard and soft domains in nanometric scales. The nanocomposite containing NSP (PU-NSP) showed better endothelial cell attachment and gene expression. The better biocompatibility of PU-NSP and PU-NSP-S was evidenced by the lower thickness of foreign body capsules in rat subcutaneous implantation. PU-NSP had the least surface degradation in vivo as demonstrated by the electron microscopy and infrared spectroscopy. This may be associated with the different surface structure. PU-NSP and PU-NSP-S showed strong bacteriostatic effects, which suggested that the nano clay in the polymer matrix may still interact with the microbes.
Journal of Biomedical Materials Research Part A 11/2011; 99(2):192-202. · 2.63 Impact Factor
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ABSTRACT: Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin. Copyright © 2011 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 10/2011; · 3.28 Impact Factor
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ABSTRACT: Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell-substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.
Biomaterials 10/2011; 32(29):6929-45. · 7.40 Impact Factor
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ABSTRACT: For surface modification and nerve regeneration, chitosan, followed by nerve growth factor (NGF), was immobilized onto the interior surface of poly (lactic acit-co-glycolic) conduits, using EDC/NHS/MES system (EDCs) and genipin (GP). Four new conduits were, therefore, obtained and named by immobilizing order-EDCs/EDCs, GP/EDCs, EDCs/GP, and GP/GP groups. The immobilized methods used were evaluated and compared, respectively. The researchers found that the EDCs- and GP-cross-linked chitosan displayed higher hydrophilic than pure poly (DL-lactic acid-co-glycolic acid) (PLGA) in water contact angle experiment, which meant the cell compatibility was improved by the modification. Scanning electron microscopic observations revealed that the GP-cross-linking of chitosan greatly improved cell compatibility while cultured rat PC12 cells were flatter and more spindle-shaped than EDCs-cross-linked chitosan. The results concerning the GP-cross-linked chitosan revealed significant proliferation of the seeded cells relative to pure PLGA films, as determined by counting cells and MTT assay. The NGF was released from the modified conduits in two separate periods--an initial burst in 5 days and then slow release from day 10 to day 40. The GP/EDCs group had the highest NGF value among all groups after the 5th day. Finally, the controlled-release conduits were used to bridge a 10 mm rat sciatic nerve defect. Six weeks following implantation, morphological analysis revealed the highest numbers of myelinated axons in the midconduit and distal regenerated nerve in GP/EDCs group. Therefore, the results confirm that GP/EDCs groups with good cell compatibility and effective release of NGF can considerably improve peripheral nerve regeneration.
Journal of Biomedical Materials Research Part A 09/2011; 99(4):576-85. · 2.63 Impact Factor
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ABSTRACT: Human gingival fibroblasts (HGF) were recently found to be a source of mesenchymal stem cells. Their behavior on a biomaterial has not been reported so far. The effect of culturing HGF on chitosan membranes on their chondrogenic differentiation was investigated in this study. HGF were first cultured on chitosan membranes and spheroid formation of HGF was observed. Next, HGF on chitosan were induced with chondrogenesis induction medium and their chondrogenic differentiation potential was expressed by assessing the expression of chondrogenesis related genes at both mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. We discovered that the chondrogenic differentiation potential of HGF could be enhanced simply by culturing HGF on chitosan membranes. Expression of neural crest and stemness genes were also analyzed by RT-PCR to evaluate the stemness and self-renewal of HGF spheroids. We found that spheroid formation helped to increase and maintain the expression of stemness genes in HGF. To understand the aspects of the chitosan membranes that induced spheroid formation of HGF, mechanical and physical properties of the chitosan membranes were examined. The migration of HGF on chitosan membranes was also monitored to speculate the process of spheroid formation. In addition, the roles of the Rho/Rho-associated kinase (ROCK) pathway and connexin 43 (Cx43) in spheroid formation were explored. Treatment of HGF cultured on chitosan with the ROCK-activity inhibitor Y27632 clearly inhibited spheroid formation, suggesting that the Rho/ROCK pathway was involved in spheroid formation. The increased Cx43 activity of HGF spheroids on chitosan indicated that the gap junction intercellular communication was regulated by spheroid formation. It was concluded that culturing HGF on chitosan may activate the Rho/ROCK pathway, which led to spheroid formation and gap junction regulation. These changes may contribute to the enhanced chondrogenic differentiation potential of HGF on chitosan.
Tissue Engineering Part A 07/2011; 18(1-2):67-79. · 4.64 Impact Factor
