| [Objective] The purpose of this research was to compare and study the proliferation and osteogenic capability of the subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia under fluid shear strees.[Methods] 1、The subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia were cultured with type-I collagenase enzyme digestion method, and purify was carried with differential attachment, the growth conditions morphology and biological characteristics of osteoblasts were observed by invert microscope; 2、Two kinds of the 4th generation osteoblast cells were identified by HE staing、ALP staining and calcific nodules of Alizarin red S staining; 3、After 1 hour and 12dynes/cm2 fluid shear stress the comparison of proliferation of two kinds of osteoblasts was carried by Cell Counting、CCK-8 absorbance method and Flow cytometry; 4、After fluid shear stress the comparison of osteogenesis ability of two kinds of osteoblasts was carried by real time PCR of OPGmRNA.[Results] 1、 There was no significant differences in the morphology of these two kinds of oteoblasts, the cells could adhere and stretch in culture bottles after 24 h, then the cells are smaller, appearing a star-shaped, triangle and short spindle, the karyon was not obvious. With the growth and differentiation, the synapse appeared in the osteoblasts surface and linked between each other, then cell fusion and multilayer growth. When the cells got into the fourth generation, the osteoblasts appeared like polygon、long spindle, the karyon was obvious, and extracellular matrix increased at the same time, then formed the cell mass combined with osteoblasts, the transmission of light got weaken, eventually calcium nodules formed, those growth characteristics conformed to the biological characteristics of osteoblast;2、The HE staining、ALP staining and calcifical nodules of Alizarin red S staining of two kinds of cells presented intense positive reaction, which were identified as osteoblasts.3、The cell counting showed after FSS the number of subchondral bone osteoblasts of temporomandibular joint condylar was significantly lowr than knee joint tibia obviously (p<0.05); CCK-8 absorbance OD showed the osteoblasts of temporomandibular joint condylar entered the logarithmic phase in the 3rd day after inoculation, and got the highest peak in the 8th day, after then entered the plateau and growth recession, while the osteoblasts of Knee joint tibia entered the logarithmic phase in the 2th day after inoculation, and got the highest peak in the 8th day, after then entered the plateau and growth recession; Flow cytometry showed the G0/G1 phase ratio of temporomandibular joint was 58.60±2.85%, while the Knee joint was 49.10±4.46%, there was statistically significant difference (p<0.05); the G2/S/M phase ratio of TMJ was 33.10±2.35%, while the Knee joint was 42.99±3.24%, there was statistically significant difference (p<0.05)4、Whether before or after FSS the OPGmRNA expression quantities of subchondral bone osteoblasts in the temporomandibular joint were lower than in the knee jiont tibial, there was statistically significant difference (p<0.05); and whether the temporomandibular joint subchondral osteoblast or knee joint tibial osteoblast, OPGmRNA expression quantities were higher than before FSS, there was statistically significant difference (p<0.05)[Conclusion] 1、The primary osteoblasts,which originated from SD rats temporomandibular joint condylar and knee joint tibia, could be cultured into the osteoblasts with the good homogeneity and the typical morphological characteristics of osteoblasts in vitro, Two kinds of cells were osteoblasts by identifing.2、In the experiment, the result of Cell Counting、CCK-8 and Flow cytometry showed the proliferation of subchondral bone osteoblasts of temporomandibular joint condylar was weaker than Knee joint tibia;3、In the experiment, the real time PCR results of OPGmRNA showed after FSS the osteogenesis ability of subchondral bone osteoblasts of temporomandibular joint condylar was weaker than Knee joint tibia in the respect of OPG, the OPGmRNA expression of two kinds of cells were higher than before FSS;4、The FSS promotes OPGmRNA expression of temporomandibular joint condylar osteoblast and Knee joint tibia osteoblast... |
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