Biological Responses Of Sox9~+ Superficial Zone Cells And Col-10~+ Deep Zone Cells In The Mandibular Condylar Cartilage To The Abnormal Dental Occlusions

BackgroundTemporomandibular joint（TMJ）condylar cartilage is sensitive to biomechanical stimulation mainly resulted from the abnormal occlusion.TMJ condylar cartilage can be divided into fibrous layer,proliferative layer,mature layer,hypertrophic and calcified cartilage layer from superficial to deep.TMJ condylar cartilage is closely related to occlusal function.In recent,our group established unilateral anterior crossbite（UAC）model in which degenerative lesions were induced in the TMJ cartilage,we also developed bilateral anterior elevation（BAE）model in which proliferative responses and matrix production were initiated.Presently,we used in vivo gene modification technology to explore the biological response of superficial and deep zone chondrocytes to abnormal occlusal stimulations of UAC and BAE.Objectives1.Using Cre-loxp gene modification technique for chondrocytes tracing in vivo in order to observe the response of Sox9⁺superficial chondrocytes and Col10⁺deep chondrocytes to UAC or BAE stimulation.2.Using DTA cell specific ablation technique to ablate Sox9⁺superficial chondrocytes or Col10⁺deep chondrocytes in order to observe the response of condylar chondrocytes to UAC or BAE stimulation.Methods1.The Sox9/Col10-Cre^ERT2mice were crossed with Rosa26/td Tomato mice to obtain Sox9/Col10-Cre ERT;R26R^{Td T}mice（n=6/5,female）.Tamoxifen was injected at the age of 6week to label,accordingly,the Sox9⁺chondrocytes and the Col10⁺chondrocytes.UAC or BAE was performed at 6-week old at which time TAM was injected.At 1-,4-,7-wks,condylar cartilage was harvested,which recorded as Sox9-Td T group and Col10-Td T group,and the changes of Td T labeled chondrocytes were observed by immunofluorescence staining.2.The Sox9/Col10-Cre^ERT2 mice were crossed with Rosa26/DTA mice to obtain Sox9/Col10-Cre ERT;R26R^DTA mice in which Sox9⁺chondrocytes and Col10⁺chondrocytes,respectively,were ablated when TAM was injected.UAC or BAE stimulation was performed at 6-week old.In Sox9 group,it was randomly divided into 3-（n=3）,7-wk（n=6）,and Tamoxifen was injected at 1-,4-,and 7-wks.In Col10 group,Tamoxifen was injected at 4-and 7-wks,then sacrificed at 7-wk,which recorded as Sox9-DTA group and Col10-DTA group.We used Micro-CT scanning,Von kossa,HE,Safranin O staining,immunohistochemical staining and TEM methods to observe the responses of condyles to UAC or BAE stimulation.Results1.Under the stimulation of UAC,the number of Sox9-Td T⁺-Col-X⁺/OCN⁺/DMP1⁺/Adipo Q⁺chondrocytes in condylar cartilage of Sox9-Td T mice was increased significantly;while under the stimulation of BAE,the number of Sox9-Td T⁺-OCN⁺/DMP1⁺/Adipo Q⁺chondrocytes was decreased.2.Under the UAC stimulation,the number of Col10-Td T⁺-OCN⁺/DMP1⁺/Adipo Q⁺chondrocytes in condylar cartilage of Col10-Td T mice was increased significantly while under the stimulation of BAE,the number of Col10-Td T⁺-Sox9⁺/OCN⁺/DMP1⁺/Adipo Q⁺cells was decreased.3.The ablation of the Sox9⁺superficial chondrocytes suppressed proliferation and differentiation of chondrocytes,leading to thinning of cartilage and cells apoptosis.Such an effect was promoted by UAC,and ruined the cartilage promotion effect of BAE.4.The ablation of Col10⁺deep zone chondrocytes resulted to aggravation of chondrocytes differentiation,cartilage thinning and cells apoptosis.Such an effect enhanced the impact of UAC on cartilage degradation and slowed down the cartialge promotion effect of BAE.Conclusions1.The Sox9⁺chondrocytes of superficial zone in mandibular condyle cartilage have the ability of proliferation and differentiation into mature chondrocytes.Ablation of the Sox9⁺superficial zone cells caused cells lack in cartilage thus dysplasia of deep zone cartilage,and enhanced the degradation lesions induced by UAC,and suppressed the proliferation effect of BAE on cartilage.2.The Col10⁺chondrocytes in deep zone have the ability of terminal differentiation.Ablation of deep zone cells ruined not only the subchondral bone formation,but also the superficial zone chondrocytes functional environment,causing deformation of the condyle and apoptosis of the superficial zone cells,which can aggravate cartilage degeneration caused by UAC stimulation and affected the proliferation activity by BAE stimulation.