牙周膜干细胞来源外泌体对正畸骨改建的影响  

Effect of exosomes derived from periodontal ligament stem cells on orthodontic bone remodeling

作　　者：雷芳草 刘原伯 LEI Fangcao;LIU Yuanbo(Department of endodontics,Hospital of Stomatology,Guanghua School of Stomatology,Sun Yat-sen University&South China Center of Craniofacial Stem Cell Research,Guangzhou 510055,China;Department of Or-thodontics,Hospital of Stomatology,Guanghua School of Stomatology,Sun Yat-sen University&Guangdong Provincial Key Laboratory of Stomatology,Guangzhou 510055,China)

机构地区：[1]中山大学附属口腔医院牙体牙髓科、中山大学光华口腔医学院、华南颅颌干细胞转化研究中心,广东广州510055 [2]中山大学附属口腔医院正畸科、中山大学光华口腔医学院、广东省口腔医学重点实验室,广东广州510055

出　　处：《口腔疾病防治》2025年第2期100-109,共10页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金青年科学基金项目(82301125、82401123);中国博士后科学基金面上项目(2024M753792)。

摘　　要：目的 探讨牙周膜干细胞来源外泌体(periodontal ligament stem cells-derived exosomes,PDLSC-Exos)在影响正畸骨改建中的作用,以期为正畸牙齿移动提供新的治疗策略。方法 本研究已通过单位伦理委员会审查批准。收集临床正畸减数拔牙的健康牙周膜组织,分离并培养牙周膜干细胞(periodontal Ligament stem cells,PDLSCs),当培养至第三代时,检测其自我更新能力和多向分化潜能。梯度离心法分离纯化PDLSC-Exos,并通过透射电镜、免疫荧光、ZetaView和纳米流式等进行鉴定。采用10μg/mL PDLSC-Exos与PDLSCs共培养诱导成骨分化(PDLSCs+Exos),评估其对成骨的影响。诱导骨髓单核细胞(bone marrow mononuclear cells,BMMs)向破骨细胞分化[30 ng/mL巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)+50 ng/mL核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)],随后加入10μg/mL PDLSC-Exos处理,以评估其对破骨细胞的影响。构建正畸牙移动大鼠动物模型(orthodontic tooth movement,OTM),并于建模前3天牙周膜局部注射50μg/mL PDLSC-Exos(OTM+Exos),2 d/次,持续14 d。Micro-CT分析牙槽骨改建、免疫组化及免疫荧光技术分析牙槽骨的破骨情况。结果 分离纯化的PDLSCs具有间充质干细胞的基本特性,且PDLSC-Exos具有细胞外囊泡的典型特征。PDLSC-Exos明显促进PDLSCs成骨分化,并促进了BMMs的破骨分化及骨吸收活性(P <0.05)。PDLSC-Exos牙周局部注射的大鼠牙槽骨改建速度明显加快,牙齿移动距离显著增加(P <0.05);免疫组化结果显示PDLSC-Exos促进破骨细胞的分化(P <0.05)。此外,免疫荧光发现PDLSCExos与破骨细胞共定位表达,说明PDLSC-Exos可能在体内促进破骨细胞的分化。结论 PDLSC-Exos促进PDLSCs成骨分化及BMMs破骨分化,并且加速了正畸骨改建的速度,从而加快了正畸牙移动。Objective To reveal the role of periodontal ligament stem cell-derived exosomes(PDLSC-Exos)in orthodontic bone remodeling,in order to provide new therapeutic strategies for orthodontic tooth movement(OTM).Methods This study has been reviewed and approved by the Ethics Committee.Healthy periodontal ligament tissues from clinical orthodontic reduction extractions were collected,and periodontal ligament stem cells(PDLSCs)were isolated and cultured.When cultured to the third generation,their self-renewal ability and multidirectional differentiation potential were detected.PDLSC-Exos were isolated and purified by gradient centrifugation and identified by transmission electron microscopy,immunofluorescence,ZetaView,and nanoflow cytometry.The co-culture of 10μg/mL PDLSC-Exos and PDLSCs(PDLSCs+Exos)induced osteogenic differentiation to evaluate the effect of osteogenesis.Bone marrow-mononu-clear cells(BMMs),promoted by osteoclast differentiation[30 ng/mL macrophage colony stimulating factor(M-CSF)+50 ng/mL receptor activator of nuclear factor-κB ligand(RANKL)],and then were treated with 10μg/mL PDLSC-Exos to assess the effect on osteoclasts.We established a rat model of OTM,and 50μg/mL PDLSC-Exos was injected locally in-to the periodontal ligament before we established the model(OTM+Exos),every 2 days for 14 days.Alveolar bone re-modeling was analyzed by micro-CT,and alveolar bone osteoclasts were analyzed by immunohistochemistry and immuno-fluorescence.Results The isolated and purified PDLSCs met the basic characteristics of mesenchymal stem cells,and PDLSC-Exos had typical characteristics of extracellular vesicles.PDLSCs-Exos significantly promoted the osteogenic dif-ferentiation of PDLSCs,and promoted the osteoclast differentiation and bone resorption activity of BMMs(P<0.05).The rate of alveolar bone remodeling in rats with local periodontal injection of PDLSC-Exos was significantly accelerated,and the tooth movement distance was significantly increased(P<0.05);immunohistochemistry results showed that PDLSC-Exos

关 键 词：牙周膜干细胞 外泌体 成骨细胞 破骨细胞 牙周膜 正畸骨改建 正畸牙移动模型 牙槽骨 

分 类 号：R78[医药卫生—口腔医学]