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Clinical bone defect caused by a variety of reasons (including trauma or tumor) is very common, and its repair has been the thorny issue of the medical profession. In recent years, the creation and development of bone tissue engineering provides a new way for the repair of bone defects. Bone tissue engineering need to meet three basic elements: seed cell growth and differentiation factors and extracellular matrix scaffold. Seed cell is the primary link and the basic elements of tissue engineering bone constructed and applied research, is to ensure that the premise of the in-depth study of the bone tissue engineering, the ideal seed cells is critical. Provide more scientific reference for the test by comparing three different sources rat osteoblast proliferation and osteogenic activity, so the choice of seed cells for bone tissue engineering. Purposes: 1. Isolated and cultured from adult rat bone marrow, periosteum and neonatal rat skull tissue in vitro rat osteoblasts mode; derived from three adult rat bone marrow, periosteum and neonatal rat skull tissue continuous observation of osteoblasts, and learn more about their biological characteristics; comparison skull periosteum, bone marrow-derived the three osteogenic cell proliferation and osteogenic activity. Methods: 1. Isolated from adult rat bone marrow, periosteum and newborn rat skull tissue of bone marrow stromal cells (marrow stromal cells, MSCs), periosteal bone cells (periosteal osteoblast, POB) and osteoblast (cranial osteoblast , COB). Primary culture, MSCs using purified by density gradient centrifugation and adherent culture method of combining culture, POB and COB were used to tissue culture method, enzyme digestion culture and enzyme digestion - Organization block joint training of law are three ways to contrast culture. Subculture process, MSCs use dexamethasone 10 -8 M, vitamin C50μg/ml and β-glycerophosphate 10mM joint vitro osteogenic induction culture; POB and COB using \speed attachment method \Test cells are the third generation of the three different sources of osteoblast cells; 2. Using inverted phase contrast microscope continuous observation of the morphological changes and biological characteristics of the three types of cells in vitro of MSCs, POB and COB; 3. MTT Law and detection of lactate dehydrogenase (lactate dehydrogenase, LDH) activity combined three cell proliferative activity; by detecting markers of bone activity of alkaline phosphatase (of alkaline phosphatase, ALP) and osteocalcin ( osteocalcin, OCN), to examine the osteogenic activity of three cells. Results: 1. Morphological observation: three different sources of rat osteoblasts were cultured to the third generation, the proliferation rate than primary speed up to about 24h after adherent, stretching, relatively uniform cell morphology in culture, fusiform or polygonal, and visible \With prolonged incubation time, the number of cells growing cells between fusion and multi-storey development, while the increased secretion of extracellular matrix and the encapsulated cells, followed by the formation of clumps, translucent gradually weakened, and ultimately the formation of calcium nodules ; 2. proliferative activity of comparison: MTT results showed MSCs begin doubled in the first 4 days into the rapid proliferative phase to day 7 proliferation rate slows down, into the plateau phase of growth; COB begin doubled in the first six days, to 9 days later proliferation rate slowed down the growth plateau; POB begin doubled in the first seven days after the proliferation slowed significantly; the LDH results show that the three types of cells in the culture to 4 days, 7 days, 14 days, MSCs LDH values was significantly higher than that of the latter two (p <0.05), the LDH value of the COB and POB in the gaps are most pronounced at day 7 (p <0.05), i.e. a COB LDH values ??significantly higher than the POB, at 4 days and 14 days gap between the two relatively small (p> 0.05), no statistically significant; osteogenesis capabilities comparison: The ALP test results show that the three types of cells in the culture to one week, two weeks, three weeks, POB ALP values ??are higher than the MSCs and COB the the 2,3 weeks POB ALP value of MSCs, COB more significant difference (p <0.05), in the first week, the MSCs the ALP value and after both gap (COB value higher than 82.39% , POB values ??higher than 109%), and the two gradually narrowing the gap with the prolonged incubation time; the OCN test results show three types of cells in culture to one week, two weeks and three weeks, MSCs osteocalcin content is always lower than the latter two (p <0.05), and the most obvious trend with incubation time extension of the widening gap between the gap of the OCN content of the COB and POB performance in the first two weeks (p <0.05), relatively small gap in 1,3 weeks (p> 0.05), not statistically significant. Conclusions: (1) using enzyme digestion - tissue block joint culture for skull periosteum-derived osteogenic cells cultured by density gradient centrifugation and adherent culture combined bone marrow stromal cells induced culture, are eligible for uniformity, activity of rat osteoblasts; (2) cultured skull periosteum-derived osteogenic cells and bone marrow stromal cells induced culture with morphological characteristics of osteoblasts, after two to three passaged kinds of cells no obvious differences in morphology; (3) rat osteoblast proliferative activity of the three different sources were: MSCs POBs the COB>; (4) of three different sources of rat osteoblasts into bone activity is as follows: POB> COB> MSCs.
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