| Bone defect is common in our clinic, and its restoration has been the thorny issue of the medical profession. recently, the creation and development of bone tissue engineering provide a new way for repairing bone defect. The bone tissue engineering includes three essential elements: seeding cell, growth and differentiation factor and extracellular matrix scaffold. In all elements, the seeding cell is the most important one. The aim of this test is to provide more scientific reference for the options of seeding cells by comparing the osteoblasts' proliferative and osteogenic activity.Objective: 1 .To establish the model of isolation and culture on the rat osteoblasts from different sources, including adult rats' marrow and periosteum and neonatal rats' cranium.2.To comprehend the bionomical characters of osteoblasts from three different sources through continuous observation. 3.To compare the proliferative and osteoplastic ability of osteoblasts from cranium,periosteum and bone marrow.Method: 1.To obtain the marrow stromal cells(MSCs),periosteal osteoblast (POB) and cranial osteoblast (COB) from adult rats' marrow and periosteum and neonatal rats' cranium respectively. When the original culture, MSCs are purified and cultured by the way of density gradient centrifugation and adherent culture, POB and the COB' culture were used three different methods including tissue culture, the culture of digestive enzymes to digest and the modified tissue culture and contrast the three different methods. In-culture process, MSCs are induced by dexamethasone 10-8M, vitamin C 50μg/ml andβ-glycerine 10mM, POB and the COB are purified and cultured by adopting "differential adhesion Law". The third generation cells are used as the test cells;2.Inverted phase contrast microscope is used to observe the morphological changes and biological characteristics of cells;3.MTT assay and LDH test are used to compare the cells' proliferative activity;4. Alkaline Phosphatase and osteocalcin are measured to compare the cells' osteoplastic activity.Result: 1. morphological Observation: when three different sources of rat osteoblasts cells were cultured to the third generation, proliferation accelerate faster than the original generation, about 24h after the training to the wall, stretching, cells were relatively homogeneous, with spindle or polygon, and that "pseudo-foot" extended. With the extension of cultivation, the number of cells is increasing, the cells are integrated and developed into multi-storey, while increasing production of extracellular matrix, and formed clumps and then, translucent gradually weakened and eventually formed nodules of calcium. 2. comparison of proliferative activity: MTT results show that MSCs in the 4th day beginning doubled into the rapid proliferation, 7th days to slow down the rate of proliferation, into a platform for growth period; COB in the 6th day beginning doubled, until the 9th day the rate of proliferation slowed, and goes into the growth platform period; POB in the 7th day beginning doubled after the speed of proliferation has slowed down; LDH results show that the cultured cells to 4th day, 7th day and 14th day, the value of MSCs' LDH Significantly higher than the latter two (p <0.05), COB and the POB ' value of LDH in 7th day exist the most obvious difference (p <0.05), in the 4th day and 14th day relative gap between the two become Smaller (p> 0.05);3.the comparison of osteogenic activity: ALP test results showed that three kinds of cells in culture to 1st week,2nd week and 3rd week, POB' value is higher than the MSCs' and COB', Of which 2nd and 3rd week, there is significant difference (p <0.05), in the 1st week, the ALP value of MSCs has a larger difference comparing with the two others(COB higher 82.39% , POB higher 109% ), with the extension of culture, and gradually narrow the gap between the two; OCN test results showed that three kinds of cells in culture to 1st week, 2nd week and 3rd week, MSCs 'value always lower than the latter two (p <0.05), and the gap is increasing with the extension of culture, the COB and POB'value in the 2nd week exist the most obvious difference (p <0.05), in 1st and 3rd week the gap become smaller (p> 0.05).Conclusion: (1) The rat osteoblasts which have good homogeneity and strong activity can be obtained through the culture or induced culture; (2) The three osteoblasts from different sources have the morphology of osteoblasts. after two generation of culture, there is no obvious difference among them in morphology; (3) the result comparison of proliferative activity: MSCs> COB> POB; (4) the result comparison of osteogenic activity: POB> COB> MSCs.
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