| Objective: The purpose of the study is to explored the feasibility of Chitosan-Gelatin/β-TCP composite as bone tissue engineering scaffolds.Methods 1.Fabrication of CS- Gel/ β-TCP Sponges The biodegradable composite scaffold was developed using beta-tricalcium phosphate(beta-TCP) with chitosan (CS) and gelatin (Gel) in the form of a hybrid polymer network via co - crosslinking with glutaraldehyde. And the sponge scaffolds were prepared by freezing and lyophilizing. The sponges exhibit a three-dimensional porous structure with 200-400μm pores.2. Cell culture 3 months old New Zealand Rabbit were chosen. the marrow stromal osteogenic cells (MSC) were collected and cultured with Dulbeccos Modified Eagle Medium (DMEM) containing 10% fetal bovine serum(FBS) .After multiplied, dexamethasone was used to promote the osteoblastic phenotype of the marrow stromal osteogenic cells(MSO). 3.Examination of the scaffolds' cytocompatibility Cell metabolism can be determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5- diphenyl-2H-tetrazolium bromide (MTT) activity located in the mitochondria and cytoplasm. According to MTT method,how the scaffolds influencing on MSC proliferation were examined . According to alkaline phosphatase (ALP -ase) method, how the scaffolds influencing on cell's ALP activity were measured. the cell -sponge constructs were measured at 6 days with light microscopy and scanning electron microscopy.4.Observation of the scaffold's biodegradability and biocompatibility in vivo 3 months old New Zealand Rabbit were subcutaneously implanted with 4 blocks of sponge scaffold in 5mm×5mm×5mm size.The implanted sponges were taken out at 4,8,12 Weeks .Histologic examination was performed with light microscopy.5. Observation of the repair of the rabbit calvarial critical size defect 12 New Zealand White rabbits were randomly divided into two groups . The round defects of diameter 15 mm were created in the parietal bone,and the same size cell-sponge constructs were used to cover these defects, but only sponges were used as controls. The rabbits were killed after 4, 8weeks. Histology and X-rays were used to evaluate healing of the defects. Result1,MSC's Multiplication and differentiation After 24 hours the MSC attached to the well as single cells and on day 3-5 formed cell groups, then formed a monolayer at the bottom of well after 1-week in vitro incubation. When bone marrow stromal cells differentiated into osteoblasts ,they formed multiple layers after another week in vitro .their ALP dyeing result was in high positive rate,at the same time there were obvious black calcium nodes.2.Result of the scaffold's cytocompatibilityWith the number of MSC increasing , MSC 's MTT activity increased .The similar MSC 's MTT activity was observed after exposure of the cells to CS- Gel/ β-TCP complex compared with controls . And MSC 's ALP activity increased as the number of MSC increased, there was no significant difference between two groups. Light and scanning electron microscopic examination indicated that seeded MSCs were firmly attached to sponge matrices and secreted extracellular matrix. 3. the scaffold's biodegradability and biocompatibility The sponge scaffolds occupied mainly by fibrous tissue retained intact for at least 4 weeks and degraded in the rough at the 12 weeks. In early period after the implantation of the sponge scaffolds ,inflammatory processes with macrophages and lymphocytes were observed ,but the processes reduceed with the sponges degraded. 4. Result of the repair of the rabbit calvarial critical size defect All the animals treated survived the experimental procedure. No inflammatory reactions, infections, or extrusions were ever observed at the implantation site. 8 weeks after operation, most area of the MSCs-scaffold complex transplanted defects was filled with newly formed bone, and new bone integrated with sponge scaffolds. In simplicial sponge scaffolds implanted defects bone format...
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