| Objective: To carry out bone tissue engineering,the osteoblasts from newborn wistar rats crania were digested by improved collagenase and cultured in vitro.After the cells were obtained,observe the biological characteristic and then co-cultured with compound scaffold materials polylactic acid -chitosan fiber (PLA-CF)/ hydroxyapatite-calcium silicate (HA-CS) in vitro. We observe the effects of PLA-CF/HA-CS on adhesion, proliferation and differentiation of osteoblasts,and to lay a foundation for the clinic uses of scaffold materials of bone tissue engineering.Methods: The rat osteoblasts were obtained from the cranium of newborn Wistar rats within 24 hours, and primarily cultured using modified collagenase digestion. In seven days osteoblasts are purified and cultured by adopting"diffenential velocity adhesion tedhnique".The cells were generated and their biological characteristic was examined by inverted phase-contrast microscope, hematoxylin-eosin staining, alkaline phosphatase (ALP) staining and mineralized nodules staining. Then the cells at passage 4 were co-cultured with PLA-CF, PLA-CF/CS and PLA-CF/HA-CS in vitro. At 3, 6 and 9 days of the culture, cell morphology was observed by inverted phase contrast microscopy. In addition, MTT assay and ALP activity test were used to observe the effects of three kinds of materials on cell differentiation and proliferation.Results: 1.Morphological observation: we found the cultured rat crania cells possessed the morphological characteristic and biological behavior of osteoblasts. The shape of cells were short spindle, triangle and polygon.2. Histological observation:A large number of blue-purple granules could be observed in the cytoplasm around the nucleus of the cultured cells with alkaline phosphorase(ALP)stain and calcium nodule formation can be observed with alizarin red stain by inverted phase contrast microscope, in accordance with the biological characteristics of osteoblasts identification standard.3. Materials and cell co-culture conditions observed: Osteoblast could increase and extend fully on three materials on the 9th day.The number of cells were most on PLA-CF/HA-CS compared with others.4.Through the testing of the cell's alksline phosphatase activity upon three materials showed that the contents of ALP increased before the nine days. The difference was signficiant between the experimental groups and the control groups (P<0.05). Meanwhile,MTT assay showed that the difference was signficiant (P<0.05) between the test groups and the control groups . The value of optical density of the different time was different(P<0.05).Conclusions:1.The cultured wistar rat crania osteoblasts consistent with osteoblast cell morphology and characterization of the identification standard.2. The results showed that osteoblasts attached on all three scaffold materials can adhere, grow, differentiate and proliferate. Furthermore, the effect of three materials on cell activity was PLA-CF/HA-CS>PLA-CF/CS>PLA-CF.The composite scaffold PLA-CF/HA-CS has a good compatibility, indicating that the material has a great potential for application in bone tissue engineering. |
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