| [Objective]1、Establish an experimental model of primary culture and subculture of SD rat’ subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia in vitro;2、Observe continuously the subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia, and discuss their biological activity;3、Further study the proliferation and differentiation of the subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia.[Methods]1、The subchondral bone osteoblasts of temporomandibular joint condylar and knee joint tibia were cultured with type-Ⅰ collagen 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 the4th generation cells were identified by HE stain、 ALP stain and calcific nodules of Alizarin red S stain;3、The comparison of proliferation of two kinds of osteoblasts was carried by Cell Counting、MTT growth curve and Flow cytometry;4、The comparison of differentiation of two kinds of osteoblasts was carried by quantitative detection of ALP、COL-1mRNA and OC.[Results]1、There was no significant differences in the morphology of these two kinds of osteoblasts, the cells could adhere and stretch in culture bottles after24h, 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 stain、ALP stain and calcific nodules of Alizarin red S stain of two kinds of cells presented intense positive reaction, which were identified as osteoblasts.3、The cell counting showed the numbers of subchondral bone osteoblasts of temporomandibular joint condylar were less than knee joint tibia obviously (p<0.05); MTT growth curve showed the osteoblasts of temporomandibular joint conylar entered the logarithmic phase in the4th day after inoculation, and got the highest peak in the8th day, after then entered the plateau and growth recession, while the osteoblasts of Knee joint tibia entered the logarithmic phase in the3rd day after inoculation, and got the highest peak in the7th day, after then entered the plateau and growth recession; Flow cytometry showed the GO/G1phase ratio of temporomandibular joint was62.90±3.15%, while the Knee joint was52.10±5.24%, there was statistically significant difference (p<0.05); the G2/SM phase ratio of TMJ was37.10±2.15%, while the Knee joint was47.90±3.24%, there was statistically significant difference (p<0.05)4、Comparing the same time, the ALP and COL-I expression quantities of subchon--dral bone osteoblasts in the temporomandibular joint were more than in the knee jiont tibial, there was statistically significant difference (p<0.05); while the OC 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); Comparing the different time, the ALP expression quantities of subchondral bone osteoblasts in the temporomandibular joint and knee jiont tibial were both gradually reduced with the time extending, there was statistically significant difference (p<0.05); while the COL-1and OC expression quantities were both gradually increased, 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;2、Two kinds of cells were osteoblasts by identifing. 3、In the experiment, the result of Cell Counting、MTT and Flow cytometry showed the proliferation of subchondral bone osteoblasts of temporomandibular joint condylar was weaker than Knee joint tibia;4、In the experiment, the quantitative detection results of ALP、COL-I showed the differentiation of subchondral bone osteoblasts of temporomandibular joint condylar was stronger than Knee joint tibia in the respect of ALP、COL-I, this was the opposite of the conclusion of proliferation; While in the respect of OC, the differentiation of subchondral bone osteoblasts of TMJ condylar was weaker than Knee joint tibia, the possible reason was that the expression of OC was positively correlated with the number and the proliferation of osteoblasts, the proliferation of subchondral bone osteoblasts of temporomandibular joint condylar was weaker than Knee joint tibia, so the OC expression of temporomandibular joint condylar was weaker than Knee joint tibia... |
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