The Effect Of Estradiol On The Proliferation And Differentiation Of Mandibular Condylar Chondrocyte

Objective:The proliferation and differentiation of mandibular condylar chondrocyte(MCC)is the foundation of the adaptive remodeling ability of condyle.Studies have shown that condylar cartilage is the target organ of estrogen,as it can be specifically bond to its receptors and produce biological effects.The mechanism of how estrogen influences the proliferation and differentiation of MCC is still unknown.Therefore,this paper establishes in vitro model of MCC using SD rat to estimate the effects of estradiol(E2)on MCC proliferation and differentiation.The aims are to find out about the biological characteristics of estradiol against MCC,and to provide evidence on the etiology of temporomandibular diseases.Methods:5 SD rats of 1-3d were used as the organization source of primary culture.The mandibular condylar cartilages were digested with trypsin and type Ⅱ collagenase to obtain the chondrocytes in vitro.Inverted microscope was used for morphological observation,and toluidine blue staining and type Ⅱ collagen immunohistochemical staining were used to identify condylar chondrocytes.The second generation of condylar chondrocytes was used for experiment,adding estradiol of 10-12 mol/L 10-9mol/L and 10-6mol/L(setting up PBS as blank control group).MTT and Flow Cytometry method were used to examine the effect of estradiol of different concentration on condylar chondrocyte proliferation and cell cycle after 24h,48h,72h.RT-PCR method was used to examine the expression change of Sox9、Col2α1、Runx2 mRN A after inducing different concentration of estradiol for 72h.Result:1.Primary generation of condylar chondrocyte have adhered after 24h in shape of triangle and polygon.About 7d,cells overgrow the bottom of the bottle and the monolayer shaped paving stone.After the passage,the cellular morphology is homogeneous,the time of adhere was shortened and growth rate was increased,overgrown the bottom of bottle after 5d.After passed to the fourth generation,the proportion of fibroblast like cells increased gradually and the cell began to dedifferentiation.When it passed to the sixth or seventh generation,the cell underwent complete dedifferentiation and presented in the shape of arborization.The growth curve of the second generation condylar chondrocyte is S-shaped,The toluidine blue staining and type Ⅱ collagen immunohistochemical staining were positive.2.After inducing estradiol of different concentration to the second generation condylar chondrocyte and culture for 24h,48h and 72h,the Estradiol of 10-12mol/L and 10-9mol/L promoted the proliferation of MCC.The 10-9mol/L group presented with the strongest positive effect,while the estradiol of 10-6mol/L has an inhibitory effect on proliferation of MCC.The promoter/inhibitory effect is more remarkable with time lapse.With the increase of estradiol concentration within 24h,the proportion of the cells in S period and G2/M period increased,while the proportion of G0/G1 period cells decreased.With the time lapse,the proportion of the S period cells decreased and remains unchanged until 72h,while the proportion of the G2/M period cell decrease gradually and the G0/G1 period cells increased gradually,as the blockage of G0/G1 period happened.3.After inducing estradiol of different concentration to cultured MCC for 72h,the expression of Sox9 mRNA in the 10-9mol/L and 10-12mol/L estradiol groups was higher than which of the control group.Among them,the expression of 10-9mol/L group higher than the control group,with statistical difference,while the 10-6mol/L group was lower than the control group with no statistical difference.The expression of Col2al mRNA in experimental groups compared with the control group,with no statistical difference.The expression of Runx2 mRNA in the 10-9mol/L and 10-12mol/L estradiol groups was lower than which of the control group.Among them,the expression of 10-9mol/L group was lower than the control group,with statistical difference,while the 10-6mol/L group was higher than the control group with no statistical difference.Conclusions:1.It’s successful and effective to apply this approach to establish culture MCC in vitro.In consideration of the primary cell purification and to prevent the cell dedifferentiation,the second generation or the third generation was the best choice.2.The effect of E2 on MCC proliferation is concentrational and chronological related.The mechanism may relate to the control of E2 to the cell cycles.3.Estradiol of different concentration influence MCC proliferation through regulation of the expression of Sox9 and Runx2.It may have functions to promote MCC maturation and hypertrophy.