咬肌肌力降低对小鼠髁突的影响

doi:10.12439/kqhm.1005-4979.2026.03.003

摘要/Abstract

摘要：

目的：

探讨咬肌肌力降低对小鼠下颌骨髁突结构及细胞生物学行为的影响，并初步分析其作用机制。

方法：

选取10只8周龄雄性C57BL/6J小鼠，随机分为咬肌注射A型肉毒毒素（botulinum toxin type A，BTX-A）组和生理盐水对照组，每组各5只。分别于第1天和第14天双侧咬肌注射BTX-A或等体积生理盐水，于首次注射后4周处死小鼠并取材。通过体视显微镜观察咬肌形态并测定其肌肉质量；采用Micro-CT分析髁突软骨下骨骨体积分数（bone volume fraction，BV/TV）、骨小梁厚度（trabecular thickness，Tb.Th）、骨小梁连接密度（connectivity density，Conn.D）及骨小梁分离度（trabecular separation，Tb.Sp）。采用苏木精-伊红（hematoxylin-eosin，HE）、阿利新蓝、抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase，TRAP）及碱性磷酸酶（alkaline phosphatase，ALP）染色观察髁突组织学变化，并结合免疫组织化学和免疫荧光检测软骨细胞凋亡、成软骨及成骨相关标志物表达。体外采用Flexcell细胞力学加载系统对C2C12成肌细胞施加不同水平（12%、6%、0%）循环牵张力（cyclic tensile stretch，CTS），收集条件培养液后与RAW264.7破骨前体细胞共培养，通过Transwell实验、免疫荧光染色及实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测破骨细胞迁移及分化能力。通过体内外实验评估BTX-A的直接毒性作用。

结果：

与对照组相比，BTX-A组小鼠咬肌体积及质量明显降低，提示咬肌无力模型构建成功。Micro-CT结果显示，BTX-A组髁突软骨下骨BV/TV、Tb.Th及Conn.D显著降低，Tb.Sp显著升高（均P<0.05），提示骨量丢失。组织学结果显示，BTX-A组髁突软骨区细胞凋亡增加，Ⅱ型胶原（collagen typeⅡ，COL2）及SRY-box转录因子9（SRY-box transcription factor 9，SOX9）表达下降；软骨下骨区TRAP阳性破骨细胞数量明显增加，而ALP及骨钙素（osteocalcin，OCN）表达无明显变化。体外实验显示，低水平循环牵张力（0% CTS）条件下，C2C12细胞条件培养液可显著促进RAW264.7细胞迁移及破骨分化，上调Mmp9、Nfatc1及Trap mRNA表达。BTX-A对破骨细胞分化及小鼠内脏组织未见明显的直接毒性。

结论：

咬肌肌力降低可通过激活髁突软骨下骨区破骨细胞，导致髁突骨质流失及退行性改变。

关键词: 肌力降低, 肌肉骨骼系统, 下颌骨髁突, 破骨细胞

Abstract:

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.

Key words: reduced muscle strength, musculoskeletal system, mandibular condyle, osteoclasts

丛荣, 曹少康, 康非吾. 咬肌肌力降低对小鼠髁突的影响[J]. 《口腔颌面外科杂志》, 2026, 36(3): 186-195.

CONG Rong, CAO Shaokang, KANG Feiwu. Effects of reduced masseter muscle strength on the mandibular condyle in mice[J]. Journal of Oral and Maxillofacial Surgery(CN), 2026, 36(3): 186-195.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2026/V36/I3/186

图/表 4

图1 注射BTX-A后小鼠咬肌及下颌骨髁突的Micro-CT与组织学分析 A.小鼠咬肌无力模型构建示意图；B.对照组与BTX-A组代表性咬肌大体照片及咬肌质量比较；C.咬肌横截面HE染色（×20）及肌纤维横截面积定量分析；D.咬肌纵截面HE染色（×20）及肌纤维长度定量分析；E. 2组小鼠术后体质量变化；F.下颌骨三维重建图、X线片及髁突软骨下骨Micro-CT定量参数比较。*表示P<0.05，**表示P <0.01，***表示P<0.001，与对照组比较。

Figure 1 Micro-CT and histological analyses of the masseter muscle and mandibular condyle in mice after BTX-A injection

图2 咬肌无力后髁突软骨区成软骨及软骨下骨区成骨的组织学评价 A.小鼠髁突HE染色结果（×40），髁突软骨各层（F：纤维层；P：增殖层；C：肥大层；H：软骨细胞层）厚度定量分析；B. Caspase3免疫组织化学染色结果（×40）及阳性面积定量分析；C.阿利新蓝染色结果（×20）；D.COL2和SOX9免疫荧光染色结果（×40）及阳性面积定量分析，白色矩形框表示定量分析取样区，tb: trabecular bone，骨小梁；E. ALP染色结果（×20）及阳性面积定量分析；F. OCN免疫荧光染色结果（×40）及阳性面积定量分析，虚线表示软骨区和软骨下区分界。**表示P<0.01，与对照组比较。

Figure 2 Histological evaluation of chondrogenesis in the condylar cartilage region and osteogenesis in the subchondral bone region after masseter muscle weakness

图3 咬肌无力促进髁突软骨下骨区破骨细胞活化及体外破骨分化 A.小鼠髁突CTSK和MMP9免疫荧光染色（×20）[下排图片（×40）为上排矩形框中的放大图]及MMP9相对阳性面积定量分析，n=5，白色三角形示破骨细胞；B.小鼠髁突软骨下骨区TRAP染色图（×20）[下排图片是上排虚线矩形框中的放大图（×40）]及破骨细胞面积定量分析，n=5，黑色三角形示破骨细胞；C.条件培养体系示意图；D. Transwell实验分析RAW264.7细胞的迁移能力（×40）及定量分析，n=3；E.MMP9和鬼笔环肽免疫荧光共染色（×40）及对MMP9阳性面积的统计学分析，n=3，白色三角形示破骨细胞；F.各组Mmp9、Nfatc1、Trap的mRNA相对表达水平，n=3。###表示P<0.001，与对照组比较；*表示P<0.05，**表示P<0.01，***表示P<0.001，与0% CTS组比较。

Figure 3 Masseter muscle weakness promotes osteoclast activation in the condylar subchondral bone and enhances osteoclast differentiation in vitro

图4 BTX-A对破骨细胞的生成无直接影响 A. TRAP染色图，下排图片（×40）为上排图片（×10）矩形框中的放大图；B.各组Ctsk、Mmp9和Trap mRNA相对表达量比较，n=3；C.小鼠主要内脏HE染色图（×20）。

Figure 4 BTX-A has no direct effect on osteoclastogenesis

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