| Mesenchymal stem cells (MSCs) are multipotent stem cells, which have potential of self duplication and differentiation. They are important member of the stem cell family and were found in the bone maroow initially. MSCs have profuse biological function, and they are able to differentiate to mesoblast and ectoderm lineage cells. MSCs can escape from the immunologic surveillance. Thus, MSCs are thought to be an ideal candidate of stem cell in tissue engineering.Silk fibroin (SF) is one compound protein which is composed of H chain (350 kDa), L chain (25.8 kDa) and P25 glucoprotein (28 kDa). The ratio of H:L:P25 is 6:6:1. The protein structure of silk fibroin can provide several sites for further biomacromolecule modification. Recent studies showed that the SF film modified by RGD([Arg - Gly -Asp])tripeptide can promote the osteoinductive capability of the biomaterial. SF can support the adherence, spread, growth and differentiation of human osteoblast, endothelial cells and epithelial cells quite well. Hydroxyapatite (Ca10(P04)6(OH)2,HA)has excellent biocompatibility because of the approximate molecular structure and Ca/P ratio to that of human bone . Accumulating studies in vivo and vitro showed that osteoblasts aggregated on the HA surface when osteoblast were co-cultured with HA material. There was no fibrous tissue interface between bone tissue and HA after HA were planted in the bone defect position. Osteoid tissue formed after HA was implanted in vivo. HA has bone conductibility and induction.In this study, SF/HA porous composites were made with SF solution and HA with different proportion. SF/HA with 400% NaCl addition, SF/HA with 600% NaCl addition, 3% SF and 6% SF porous materials were chose after initial screening about their basic capabilities. Furthermore, four bilayer porous mterials were made from above mentioned four kinds of porous materials. This study mainly consists of two parts. In the first part, we focused on the cytocompatibility of the SF and SF/HA materials. The murine mesencymal stem cells C3H10T1/2 were seeded on the SF and SF-HA films. The cells were cultured for five days and the cells proliferation ability were detcted by MTT assay every day. Moreover, C3H10T1/2 cells were seeded on four kinds of porous materials and the proliferation ability of the cells was detected by MTT assay every day during the five days of culture proceed. Scanning eletron microscope was used to detect the adherence and proliferation of the cells on the porous materials. The result revealed that the proliferation of the cells on SF films was better than that on SF/HA films. Both of the cell proliferation capabilities on the two film groups were better than that on the control group. The cell proliferation ability of 600% SF/HA porous material group was better than that of 400% SF/HA group. However, there was no difference between the two SF porous materials groups. Cells in the four porous materials groups could adhere on the materials and proliferate well. The extracellular matrix secretion was observed by the scanning eletron microscope. The cells could adhere well to the bilayer porous materials. In addition, the two layers of the bilayer porous materials sticked together well during the culture procedure.In the second part, we focused on the osteogenic induction effect of the SF and SF/HA porous materials with human placenta-derived mesenchymal stem cells (PMSCs). Human PMSCs were primary cultured and identified for their typical morphology, phenotype and differentiation capacities. PMSCs were seeded on the four kinds of porous materials separately. The first group included four kinds of porous materials with PMSCs cultured in the osteogenic induction medium. The second group consisted of the same four porous material with PMSCs cultured in the non-osteogenic induction medium. The third group included the four materials only without PMSCs as control. They were cultured for fifteen days. The supernatant of the cells culture were collected every three days during the culture and the alkaline phosphatase (ALP) activity were detected. After fifteen days of culture the ALP activity of the cells were detected for each group. The result showed that 600%SF/HA and 6%SF could support the PMSCs differentiate to osteocytes better than the other two kinds of porous materials. There was no obvious difference of ALP activity between the four materials in the none-induction group and control group. Therefore, it is consider that the SF/HA alone can not induce the osteogenic differentiation of human PMSCs in vitro.In summary, the four types of porous materials have good cytocompatibility in vitro. 600% SF/HA and 6%SF porous materials have higher efficiency to support the osteogenic differentiation of PMSCs compared to the other two kinds of materials individually. Thus, the above two kinds of materials are chose as ideal biomaterial candidates for in vivo experimental studies, which will provide further theoretical basis for their applications in tissue engineering.
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