The Role Of ACVR1-mediated BMP Signaling On Condylar Cartilage In Postnatal Mice

Background:Mandibular condylar cartilage?MCC?plays an important role of mandibular growth and joint activity.However,there are still few studies on the genetic,cellular and molecular mechanisms of MCC morphogenesis and development.Genetic studies in mice have shown that bone morphogenetic protein?BMP?signaling pathway is involved in the regulation of cartilage growth and development.BMP receptors are composed of type I and type II receptors,of which type I receptor is the main determinant of specific signal transduction.ACVR1?activin A receptor,type I,?is one of type I BMP receptors.In recent years,the role of ACVR1 in growth plate cartilage has been gradually reported,and the conditional knockout of Acvr1?Col2a1-Cre?in growth plate cartilage of mice has no obvious abnormalities compared with wild-type littermate.Due to the differences in embryogenesis,tissue structure,and response to growth factors between growth plate cartilage and condyle cartilage,ACVR1 may have a different effect on growth and development of condyle cartilage.In vitro experiments of our research group confirmed that ACVR1 can inhibit the proliferation of mouse condylar chondrocytes and promote the differentiation of mouse condylar chondrocytes into chondrocytes.However,due to the complex and changeable environment in the body,no literature has reported the role of ACVR1 in the occurrence and development of MCC.Objectives:To observe the effects of type I BMP receptor ACVR1 on the condylar cartilage of postnatal mice and explore its effects on the morphology,cell proliferation and differentiation of the condylar chondrocytes,which will provide a basis for the etiology and treatment of cartilage-related diseases of the temporomandibular joint?TMJ?.Materials and methods:Using the Cre-LoxP recombinase system,a C57BL/6J mouse model of the Acvr1gene conditionally knockout in condylar chondrocytes was constructed and genetically identified.By mating female and male mice with genotypes Acvr1 ^fx/fx;RS/RS and Acvr1^+/-;Osterix?+?/?-?respectively,the offsprings with the genotype of Osterix-Cre?+?/?-?;Acvr1 ^fx/-;RS/+were designated as the experimental group,and Osterix-Cre?+?/?-?;Acvr1 ^fx/+;RS/+mice as the control group.Male and female mice of postnatal day 0?PN0?,PN21 and PN42 were collected.To determine the specificity of gene knockout,X-gal staining was used to detect Cre activation at PN0,and immunohistochemical staining?IHC?was used to detect the localization of Osterix at PN21.The morphology of the condyle was examined by micro-CT at PN21.The histology of the condylar cartilage was examined at PN21 and PN42 by HE and toluidine blue staining.Immunohistochemical staining for PCNA and type X collagen?Col X?was used to detect the proliferation and differentiation of condylar chondrocytes at PN21 and PN42,respectively.Results:X-gal staining and IHC results showed that the mouse model of Acvr1 gene deletion was successfully constructed.At PN21,the width and the length of the condylar cartilage of the male mice in the experimental group were significantly decreased?P<0.05?,and the width of condylar cartilage of the female mice was decreased?P<0.05?,compared with the corresponding controls,however,there was no difference in the length of condylar cartilage of the female mice.At PN21,the morphology of the condylar chondrocytes and the histological structure of condylar cartilage in the male and female experimental groups were not significantly different from those in the control group.However the chondroblastic zone?Ch?in the intermediate part of the condylar cartilage in the male experimental group showed a thicker trend?P=0.06?.The hypertrophic chondrocyte zone?Hy?in the posterior part of the condylar cartilage in the female experimental group showed a thinner trend?P=0.11?.At PN42,the chondrocytes in experimental group were disordered in both male and female mice.Specifically,the Hy of the anterior part and the total thickness of the Ar,proliferating zone?Pr?and Ch in the experimental male mice were significantly thicker,compared with the control group?P<0.05 and P<0.01,respectively?.The thickness of the Ch in the anterior part showed a thicker trend in experimental male group,compared with the control group?P=0.083?.In female group at PN42,Ch and Hy in the posterior part of condylar cartilage were significantly thicker than control group;the total thickness of the Ar,Pr and Ch in the intermediate part?P=0.16?and the thickness of the Ar and Pr in the posterior part?P=0.051?of the condylar cartilage showed thicker trends,compared with the control group.PCNA IHC staining results showed that at PN21,the number of PCNA positive cells in the Ch of male experimental group was significantly higher than that in the control group?P<0.05?,with an increasing trend of the number of PCNA positive cells in Hy in the male experimental group,compared with the control group?P=0.13?.However,the mean optical density of type X collagen in Ch and Hy of male experimental group was not statistically different from that in the control group.In females,the number of PCNA positive cells in each layer of the condylar chondrocytes of in the experimental group showed no difference from that in the control group;the mean optical density values of type X collagen in the condylar Ch and Hy of female experimental group showed no difference compared with the control group.At PN42,the number of PCNA positive cells in the condylar cartilage of male and female mice?count together?in the experimental group was significantly higher than that in the control group?P<0.05?.The mean optical density of type X collagen in the Ch of male experimental group was significantly higher than that in the control group?P<0.05?,but the mean optical density values of type X collagen in female experimental group were not significantly different from those in the control group.Conclusions:In the Osterix-Cre conditional gene knockout model,ACVR1 did not regulate the morphology and structure of the condylar cartilage before weaning stage in mice;ACVR1 in the condylar chondrocytes regulated the structure of the condylar cartilage in an age-dependent and gender-dependent manner;ACVR1 affected the cell morphology and tissue structure of condylar cartilage by inhibiting the proliferation of the chondrocytes and the differentiation of hypertrophic chondrocytes from chondroblasts.