| ObjectiveAlthough the study of the construction of the bony tissue engi-neering in vitro is adequated , the applied extracellular scaffold has much defection compared with the natural bone tissue. Recently , a new kind of biological material , named bony Acellular Extracellular Matrix - AECM, was invented, which is natural extracellualr matrix without cells structure conducted by the physical and chemical meth-od, and it is similar to the natural bone tissue. Some experts described the characteristic on the bony AECM and confirmed it was a good ex-tracellullar scaffold , but up to date , there have not the report in de-tail about the coculture of the cell in the bony AECM . Osteoblast is the essential cell in the osteosis,and in vitro ,it have the features of easy obtaining and culture , rapid magnification. Osteoblast is the per-fected seed cell for the bony tissue engineering, but there have not the completed report about the osteogenic characteristic. Our study take the long-bone--graft osteoblast as the seed cell and coculture with the bony REAECM in vitro in order to provide the bases to the choice of the seed cell of the bony tissue engineering and the experimental data of the construction of the tissue engineered bone in vitro.Materials and MethodsFifteen Wistar rats weighted by 0. 18 - 0. 2 kilogram were pur-chased from Animal Department of China Medical University ( Liaon-ing, China). Dulbecco's Modified Essential Medium (DMEM) was purchased from GIBCO/BftL and fetal bovine serum (FBS) was pur-chased from TDB Biological Production Co. Ltd (Tianjin,China). The trypsin was purchased from DIFCO. Polyclonal rabbit antibody against collagen type I was obtained from Wuhan Bosten biological production Co. Ltd (Hubei,China). Preparation of the bony REAECMAnesthetized by 1 % sodium pentobarbital, two middle segment of femurs from five wistar rats were taken down. Getting ride of the peri-osteum and being cut into small pieces , the bones were washed by PBS. Bone patches were put into bottle containing 0.05 M Tris - HCL (pH7. 4) buffer and proteinase inhibitors (0. 1μg/ml aprotinin,0. 5μg/ml leupeptin,0. 6μg/ml pepstatinA). After four days, the sam-ples were submerged in Tris - HC1 buffer containing 3% Triton x -100 and proteinase inhibitors and vibrated at 4癈 constant tempera-ture for seven days. Then, distilled water washed bone patches. Next-ly, the samples were digested by DNase and RNase at 371 for 12 hours. Then the samples were immersed into Tris - HC1 buffer contai-ning 3% Triton x - 100 for seven days again and washed by distilled water again. The samples were put into Von Ebnel liquid for 30 days for decalcification and were stored in the PBS containing 4 x104 unit Getamycin . lastly , the AECM was collected in the beaker and bro-ken into pieces. Then the AECM was centrifugalized and deposited the remains in the wells of 24 - multiwell plate and let it natural deposit and being dried. Then the bony REAECM were put into the alchohol for 48 hours and rayed by the x梤ay for three days. Some were stored for the coculture , others were observed by HE staining and ScanningElectron Microscope.The culture and observation of osteoblast of Wistar rat in vitroThe middle section of femoral bones dissected from ten wistar rats were washed clearly in PBS and cut into pieces, then digested with 0. 25% trypsin/EDTA (DIFCO) for 30 min x5 times in the 37t water pool and overnight in the refrigerator at 4癈 temperature. Next morn-ing ,the bone masses were digested again with 0.25% trypsin/EDTA (DIFCO) for 2 hours in 37癈 water pool ,then submerged in DMEM (DMEM; Gibco) supplemented with 10% FBS and 1% penicillin/ streptomycin under standard cell culture conditions (that is, 37癈, humidified 5% C02) and changed the DMEM alternate day. The oste-oblasts at the forth generation were used for cellular growth curve of the thiazolyl blue colorimetry Hematoxylin- eosin Mallory-heiden-hain Alizarin Red-S Calcium梒obalt Alkaline Phosphatase - Im-munohistochemical collagen type I staining...
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