| TMJ(Temporomandibular joint)plays an important role in craniofacial growth and function. Unlike the long bones and joints, When the TMJ gets trauma, its intra-articular environment changes could easily caused MCC(Mandibular condylar cartilage) pathological changes resulting in temporomandibular joint osteoarthritis occurs. This process can let the MCC loss growth ability and accelerate EO(Endochondral Ossification) [1] process and condylar pathological calcification. In normal cartilage, type II collagen is 60% in the total dry weight of cartilage. The cracking and collapse of Col II will directly lead to the structure of cartilage destroyed, while accelerating inflammation excitation leads to a vicious cycle of disease, eventually, the cartilage calcification. MMP-13(Matrix metalloproteinases-13)could potent degrade proteins which is widely present in ECM(Extracellular matrix)[2].MMP-13 is 10 to 30 times in destruction of Col II than other collagenase [3]. VEGF(Vascular endothelial growth factor) can promote cartilage vascularization and production MMP-13, while inhibiting its inhibitors TIMP-1 and TIMP-2 expression and accelerate the process of OA [4]. Hyaluronate(Hyaluronic Acid, HA) can effectively alleviate the OA process [5], TGF-?1 can accelerate the process of OA by down-regulated the activity of mir-140 [6]. This task taken the MCC and long bone cartilages tissue cultured in vitro to test the expression of VEGF and MMP-13 in MCC calcification process which compare with long bone, then we added appropriate concentration of HA or TGF-?1 in the medium to observe the situation of calcification while detecting the VEGF/MMP-13 expression situation. This task could provide us some theoretical basis for clinical study and diagnoses of TMJOA.Method: The experiments were performed using a small tissue cultured in vitro. Experiment one: In a clean bench, decapitated the 3 days of newborn mice under the stereomicroscope to get their MCC and long bone cartilages(tibia head, femoral head). The cartilages were set two group randomly,one is control group,which were fixation directly. The other cartilages were set as medium control group and cultured 6 weeks in vitro in the blank IMDM medium. We use a stereoscopic microscope to observe the morphological changes,HE staining to observe organizational structure changes,alizarin red and alkaline phosphatase staining(ALP) staining to analyze the situation of tissues calcification. We use IPP6.0(Image-Pro plus6.0) software to meter the change of cartilage area and use the immunohistochemical and positive cell counting to test the expression of VEGF/MMP-13 in MCC and long bone cartilages of medium control group; Experiment two: Only MCC to be studied. Basis on experimental one, a suitable concentration of HA was added in IMDM culture medium in HA group. After 6 weeks culture, we observe the situation of MCC calcification and use the same methods to test the expression of VEGF/MMP-13.All the results were compared with medium control group; Experiment three: The appropriate concentration of TGF-?1 was added to the medium. After 6 weeks culture, we observe the situation of MCC calcification and use the same methods to test the expression of VEGF/MMP-13. All the results were compared with medium control group. All data were analyzed statistically by SPSS software to study of the expression of VEGF and MMP13 in mandibular condylar cartilage calcification.Results: 1.The MCC was calcification after 6 weeks culture in vitro, VEGF and MMP-13 expression was increased.The MCC cultured in blank IMDM medium( medium control group) were calcification after 6 weeks. We can observe an arched density and light blocking area in the cartilage under the stereomicroscope. ALP and alizarin red staining showed that compared with previous culture(control group), the light blocking area occur calcification. HE staining showed: After 6 weeks cultured, condylar cartilages get thinner than before and cartilages lack unity and coherence, partially chondrocyte occur hypertrophic differentiation, ECM occurs basophilic arisen. Immunohistochemistry and the positive cell count analysis showed: the number of positive cells of VEGF and MMP-13 were increased after 6 weeks cultured in vitro, VEGF and MMP-13 were expression in calcification area of cartilage ECM. This experiment proves that MCC calcification accompanied the expression of VEGF and MMP-13 in the ECM and the positive cells increasing.2. Long bone cartilage was not observed calcification and the cartilage surface area was increased.Long bone cartilage(tibia and femoral head cartilage) after 6 weeks cultured in vitro was not observed light blocking area under the stereomicroscope. ALP and alizarin red staining results show that long bone cartilage after cultured in vitro without calcification. HE staining: The layers of long bone cartilage after 6 weeks cultured in vitro still clear and the ECM area was not checked basophilic arisen. Immunohistochemistry and the positive cell count analysis showed that after 6 weeks cultured in vitro, long bone cartilage VEGF and MMP-13 positive cells increased. Cartilage surface area measurement results show that after 6 weeks cultured in vitro, long bone cartilage surface area increased. The results tell us different of condylar cartilage, long bone cartilage could continue to grow without calcification in the same culture environment.3. The MCC of HA group didn't calcification and the positive cells of VEGF/MMP-13 were restrained.After 6 weeks cultured in vitro, all condylar cartilages of HA cannot observe light blocking area occur under the stereomicroscope. ALP and alizarin red staining did not check the condylar cartilage calcification. HE staining results showed that the thickness of condylar cartilage after culture was increased and the layers of condylar cartilage could be identify, meanwhile the ECM basophilic arisen negative. The cartilage surface area measurement shows us the surface of MCC was increased. All the results show us that the MCC of HA group didn't calcification. The results of immunohistochemistry show us that VEGF/MMP-13 was negative in the ECM of MCC from HA group. The positive cells counting results showed us that the positive cells of VEGF or MMP-13 were decrease in MCC of HA group.4. TGF-?1 can accelerate the calcification and increase VEGF/MMP-13 expression.After 6 weeks cultured in vitro, the light blocking area of TGF-?1 group condylar cartilage was observed and larger than the medium control group. ALP and alizarin red staining results show that the calcification of TGF-?1 group condylar cartilage was more serious. HE staining shows us that condylar cartilage differentiation hypertrophy and ECM area basophilic arisen serious, while cartilage cell layers chaos even part of the cartilage cell fractured could telling us that TGF-?1 could promote the MCC calcification. The results of immunohistochemistry show us that the calcification area can test the VEGF/MMP-13 expression. The positive cells counting results showed us that the positive cells of VEGF or MMP-13 were increase in MCC of TGF-?1 group compare with medium control group.Conclusion: 1. MCC is different from long bone cartilage, after 6 weeks cultured in vitro, due to the change of the surrounding environment, condylar cartilages show the characteristics of the secondary cartilage occur pathological change. Long bone cartilage is primary cartilage, its growth is different to affect by the surrounding environment change and could continue to grow without calcification,so the long bone cartilage was gave up in experiment two and three, only the condylar cartilage with calcification to be studied. 2. In the process of MCC calcification, the calcification area of MCC could test the expression of VEGF/MMP-13. 3. In vitro,MCC of HA group didn't calcification, only little positive cells of VEGF and MMP-13 expression,the positive cells of VEGF and MMP-13 decreased. 4. In vitro, TGF-?1 could accelerate the MCC calcification and the calcification area of MCC could test the expression of VEGF/MMP-13. The positive cells of VEGF and MMP-13 increased. |
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