| Part.1 Design and fabrication of novel structure PLGA/collagen hybrid scaffoldsObjective:To design and fabricate PLGA/collagen scaffolds with novel structures for the sake of future application in the research of regeneration medicine.Methods:Prepare the PLGA/collagen hybrid scaffolds with different structures by forming collagen microsponges in the openings or the sides of a PLGA knitted mesh. 3 kinds of structures were achieved using special designed moulds. (1) THIN: collagen micro-sponge formed in interstices of PLGA mesh; (2) SEMI:collagen micro-sponge formed on one side of PLGA mesh; (3) SANDWICH:collagen sponge formed on both sides of PLGA mesh. Surface and cross-section morphology were observed by scanning electron microscopy. Pore sizes were calculated with image analyzing software. Water uptake and swelling ratio was also evaluated.Results:THIN, SEMI and SANDWICH PLGA/collagen scaffolds with designed structures were successfully fabricated. The surface pore sizes in HIN, SEMI and SANDWICH scaffolds were 136.4±11.8μm,142.9±15.3μm and 139.6±12.5μm respectively. The swelling ratio of SEMI and SANDWICH scaffolds were 12.9±1.71 and 13.6±2.3, much higher than THIN scaffold which was 4.4±0.4 (P<0.001).Conclusion:THIN, SEMI and SANDWICH PLGA/collagen scaffolds with novel structures were prepared. The micro-structure and pore sizes, increased collagen micro-sponges were expected to facilitate cell adhesion, tissue accommodation and regeneration. Part.2 Effect of novel structure PLGA/Collagen hybrid scaffolds on chondrogenesis in vitroObjective:To investigate and compare the effect of THIN, SEMI, SANDWICH PLGA/Collagen hybrid scaffolds on the chondrogenesis in vitro.Methods:Bovine articular chondrocytes (BACs) were isolated, cultured and examined by phase contrast microscopy. P2 BACs were seeded onto the scaffolds and seeding efficiency was investigated. (1) THIN/BACs:BACs were seeded onto THIN PLGA/Collagen scaffolds; (2) SEMI/BACs:BACs were seeded onto SEMI PLGA/Collagen scaffolds; (3) SANDWICH/BACs:BACs were seeded onto SANDWICH PLGA/Collagen scaffolds. Cell growth on the surface and inside the scaffolds were observed by SEM. Quantification of DNA and GAG was carried out after the samples were cultured in vitro for 1 week. Real-time PCR was done to evaluate the expression of type I collagen, type II collagen and Aggrecan mRNA. Compare was done between the THIN, SEMI, SANDWICH group, BACs cultured in flasks for 1 week and natural cartilage.Results:BACs were successfully isolated and multiplied. The seeding efficiency of BACs on SEMI and SANDWICH group were 87.8±1.6% and 95.6±2.2% respectively, much higher than THIN group (P<0.05). Homogenous cell distribution and active cell growth were achieved throughout the scaffolds when checked by SEM. DNA volume in SEMI and SANDWICH groups were significant higher than THIN group (P<0.05), while no difference in GAG volume and lower than those of natural cartilage. The typeⅡcollagen and Aggrecan mRNA expression showed no difference between 3 groups.Conclusion:Excellent biocompatibility was shown in PLGA/Collagen hybrid scaffolds to BACs. The novel design of SEMI and SANDWICH increased the cell accommodation capability by building thicker collagen micro-sponges on the PLGA knitted mesh. The seeding efficiency of BACs on SEMI and SANDWICH scaffolds were greatly improved. Hyaline cartilage-like ECM excretion was enhanced in SEMI and SANDWICH scaffolds much more than THIN group. Part.3 Effect of PLGA/Collagen novel structure hybrid scaffolds on chondrogenesis in vivoObjective:To investigate and compare the effect of THIN, SEMI, SANDWICH PLGA/Collagen hybrid scaffolds on the chondrogenesis in vivo.Methods:P2 BACs were seeded onto 3 kinds of PLGA/Collagen hybrid scaffolds, and after cultured for 1 week in vitro, all the samples were transplanted subcutaneously in the dorsum of nude mice. (1) THIN/BACs:BACs were seeded onto THIN PLGA/Collagen scaffolds; (2) SEMI/BACs:BACs were seeded onto SEMI PLGA/Collagen scaffolds; (3) SANDWICH/BACs:BACs were seeded onto SANDWICH PLGA/Collagen scaffolds; (4) Control:empty PLGA/Collagen scaffold of each group was selected randomly. Samples were harvested at 2,4 and 8 weeks after transplantation and the thickness were valued. Quantification of DNA and GAG was carried out and expression of type I collagen, type II collagen and Aggrecan mRNA were evaluated by Real-time PCR. Histological and immune-histological staining was done for the transverse and cross-section of the samples. The biomechanical properties of all the 3 groups were also examined.Results:Gross view of THIN/BACs, SEMI/BACs and SANDWICH/BACs samples were cartilage-like and ivory white when harvested at 2,4,8 weeks after transplantation, and the original shapes were retained after 8 weeks. The thickness of the new formed cartilage in SEMI/BACs and SANDWICH/BACs was 1.66 and 1.73 times higher than that of THIN/BACs (P<0.05). DNA volume in 3 groups were quite similar while the GAG quantity perμg DNA was much higher in SEMI/BACs and SANDWICH/BACs groups (P<0.05), about 60.1% and 52.1% of that of natural cartilage. Real-time PCR revealed that the expression of type II collagen and Aggrecan mRNA increased with the transplantation time while the expression of type I collagen mRNA decreased. Histological and immune-histological staining showed a homogenous cell distribution and abundant hyaline cartilage-like ECM excretion. When compared to natural cartilage, mechanical strength of the engineered cartilage in SEMI/BACs and SANDWICH/BACs groups reached 54.8%,49.3% in Young's modulus and 68.8%,62.7% in stiffness respectively.Conclusion:Excellent biocompatibility was shown in PLGA/Collagen hybrid scaffolds after being transplanted in nude mice. The novel design of SEMI and SANDWICH scaffolds enhanced the chondrogenesis in vivo compared to THIN scaffold. No significant difference was observed between the effects of chondrogenesis by SEMI or SANDWICH scaffolds. The engineered cartilages are quite similar to the natural articular cartilage in the aspects of chemical compositions and biomechanical properties.
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