Experimental Study On The Effect Of Simulated Microgravity On Osteoblasts And Chondrocytes Seeded On The Collagen-Chitosan-β-TCP Composite Scaffold In Vitro Culture

Articular cartilage defects is one of the orthopedics clinical problems, articular cartilage autotransplantation limited by its shortcomings such as sources, trauma, the high cost. Articular cartilage allograft transplantation difficult to rule out the possibility of endogenous immune problems. Along with the development of tissue engineering, using artificial regeneration organizations to repair articular cartilage defects, providing new ideas and methods to solve the problem . Although the articular cartilage tissue engineering research has made great progress, there is still a large number of issues to be resolved. Particularly cartilage and subchondral bone tiered, fracture, the host integration, and the long-term effects of articular cartilage repairing.Objective: Collagen, chitosan andβ-tricalcium phosphate are compounded organically to building layered gradient composite, in order to imitate the hierarchical structure of articular cartilage and increase the mechanical strength of scaffold. The osteoblasts were induced from rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a stimulated microgravity environment respectly, The effects of the composite scaffold on the cell adhesivity, proliferation and morphological changes were observed, to evaluate the feasibility of composite material as articular cartilage tissue engineering scaffolds and observe the effectiveness of simulated microgravity on cells and constructs.Methods: Put the cells and scaffold as experimental object, The experiment were given to two groups: the general culture and microgravity culture, the general culture as a control. MTT assay subgroups: five time groups: first day, third day, fifth day, seventh days,ninth day. Five parallel hole / group, 50 holes totally.1.BMSCs composite materials: the rabbit primary BMSCs were cultured by whole bone marrow adherent screening method and induced to osteoblasts,the result improved by detecting the activity of alkaline phosphatase and formation of calcium nodule after three weeks culture,then the BMSCs were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type I immunohistochemical staining after four weeks.2. Chondrocytes composite materials: the New Zealand immature rabbit primary chondrocytes were cultured in vitro amplification, the second and third generation cells were improved by S-100 immunocytochemistry staining ,then the first four generation chondrocytes were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type II immunohistochemical staining after four weeks.Statistical analysis: MTT assay, the same time point of comparison between the two groups using two independent samples t-test .the same group at different time points compared using the completely randomized design of single-factor analysis of variance (One-way ANOVA); two overall comparison between the two groups using analysis of variance of repeated measures data. a= 0.05 is the level for the statistical test.Results: 1. Cell morphology: BMSCs were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 24 h with its fusiform shape in majority and polygonal in minority. The cells covered culture flask about one week . After be induced, the cells grew slowly and changed its form into more large shape ,then overlapped one another. There were a mount of round or oval calcified nodules among cells two weeks later and distributed in radiat, the cells' Alkaline Phosphatase staining was positive and Von Kossa method staining of calcified nodules was positive. The chondrocytes were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 5h with its triangle shape in majority and polygonal in minority. The cells covered culture flask about one week with "paving stones" appearance. The S-100 immunocytochemical staining of chondrocytes ware positive.2. Cell / scaffold microscopy: After dropping cells suspension in the wet pretreatment scaffold, the scaffold expansion rapidly revealed that the cells spread in it. The result of inverted microscope revealed that the scaffolds were opaque and a large number of osteoblast and chondrocytes adhered the adjacent material. 3. MTT assay: After osteoblasts were seeded onto the scaffolds , There was a significant difference between two groups at different time points (P <0.05) except the first day (P = 0.706), and group-factors was also statistically significant (P = 0.000).After chondrocytes were seeded onto the scaffolds , There was a significant difference between the two groups at different time points (P <0.05), and group-factors was also statistically significant (P = 0.000).4. Scaning Electron Microscope: After cultured 7 days, the cells agglomerate adsorbed on the surface and pore walls of scaffolds, linked with each other by cell processes or stretched out pseudo-foot attached to the pore wall. Discussion from the middle of scaffold, there were a small number of cells attached to pore wall.5. HE staining and immunohistochemical staining: Osteoblasts and scaffolds: After 4 weeks, there were a large number of cells with spherical shape at the surface and a small amount of cells overlapped in it , around which deposited much extra cell matrix .The typeⅠcollagen immunohistochemistry show positive response within the cytoplasm. Chondrocytes and scaffolds: After 4 weeks,there were a large number of cells with round shape at the surface and a small amount of cells overlapped in it, around which deposited much extra cell matrix . The typeⅡcollagen immunohistochemistry show positive response within the cytoplasm.Conclusion: 1. We develope successfully New Zealand rabbit primary chondrocytes and BMSCs, and successfully induced BMSCs to osteoblasts, which provided sufficient quantity and better biological properties cells resource for further experiment. 2. Collagen / chitosan /β-TCP layered gradient composites simulate the hierarchical structure of normal articular cartilage, which can provide three-dimensional growth environment for cells with its better compatibility. 3. Simulated microgravity from rotating wall vessel bioreactor can promote osteoblasts and chondrocytes grow in high density in scaffold, and promote the secretion of extracellular matrix, which improve the quality of tissue-engineered articular cartilage in vitro and open a new way for the articular cartilage tissue engineering research.