咬肌肌力降低对小鼠髁突的影响OA

Objective:To investigate the effects of reduced masseter muscle strength on the structure and cellular biological behavior of the mandibular condyle in mice and to preliminarily analyze the underlying mechanism.Methods:Ten 8-week-old male C57BL/6J mice were randomly assigned to a botulinum toxin type A(BTX-A)injection group and a saline control group(n=5 per group).BTX-A or an equal volume of saline was injected bilaterally into the masseter muscles on days 1 and 14,and all animals were sacrificed 4 weeks after the first injection,and tissues were harvested.Masseter muscle morphology was observed under a stereomicroscope,and muscle weight was measured.Micro-CT was used to analyze bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular connectivity density(Conn.D),and trabecular separation(Tb.Sp)in the condylar subchondral bone.Histological changes in the condyle were examined using hematoxylin-eosin(HE),Alcian blue,tartrate-resistant acid phosphatase(TRAP),and alkaline phosphatase(ALP)staining;immunohistochemistry and immunofluorescence were performed to detect chondrocyte apoptosis and the expression of chondrogenic and osteogenic markers.In vitro,C2C12 myoblasts were subjected to cyclic tensile stretch(CTS)at different magnitudes(12%,6%,and 0%)using a Flexcell mechanical loading system.Conditioned media were collected and co-cultured with RAW264.7 pre-osteoclast cells.Osteoclast migration and differentiation were evaluated by Transwell assay,immunofluorescence staining,and real-time quantitative polymerase chain reaction(RT-qPCR).The direct toxicity of BTX-A was assessed both in vivo and in vitro.Results:Compared with the control group,the masseter muscle volume and weight were significantly reduced in the BTX-A group,indicating successful establishment of the masseter weakness model.Micro-CT analysis demonstrated significantly decreased BV/TV,Tb.Th,and Conn.D,along with significantly increased Tb.Sp in the condylar subchondral bone of the BTX-A group(all P<0.05),indicating bone loss.Histological analyses revealed increased chondrocyte apoptosis and decreased expression of collagen typeⅡ(COL2)and SRY-box transcription factor 9(SOX9)in the condylar cartilage of the BTX-A group;the number of TRAP-positive osteoclasts in the subchondral bone was significantly increased,whereas the expression of ALP and osteocalcin(OCN)showed no significant changes.In vitro experiments showed that conditioned media from C2C12 cells under low-level mechanical stretch(0%CTS)significantly promoted RAW264.7 cell migration and osteoclast differentiation and upregulated the mRNA expression of Mmp9,Nfatc1,and Trap.BTX-A showed no obvious direct toxicity to osteoclast differentiation or visceral tissues of mice.Conclusion:Reduced masseter muscle strength can lead to bone loss and degenerative changes in the mandibular condyle by activating osteoclasts in the condylar subchondral bone.