Periostin在纯钛种植体表面促进仿生性牙周膜再生的作用机制研究

Osseointegrated implants have been widely utilized in restoring oral functions for patients with dentition defects, but lacking biomimetic periodontal ligament like natural teeth, osseointegrated dental implants demonstrate higher sensitivity to complicated biomechanical and microbial environment in the mouth. Although many attempts have shown the possibility to construct periodontal ligament (PDL) around implants, how to regenerate biomimetic PDL with elaborate structures and well-arranged fibers still proves to be a hard-nut, which involves fine intracellular tuning, interaction between cells and extracellular matrix and stress environment. As a matricellular protein, periostin plays a vital role in dental development, collagen fibrillogenesis, bone formation and tissue healing, and maintains dental tissue integrity under mechanical loading. We presume that periostin can promote periodontal ligament stem cells(PDLSCs) to regenerate periodontal ligament complex on the surface of titanium implants, and have previously observed its effect on PDLSCs osteogenic differentiation. This project aims at the critical problems of osseointegrated implants that lack biomimetic PDL and attempts to clarify the mechanism of periostin in regeneration of biomimetic PDL around implants by in vitro and in vivo studies using techniques of cell and molecular biology , stem cell sheet and biomaterial surface treatment, which will pave the way for the birth of real biomimetic dental implants that serve long and well for patients with tooth loss.

骨结合种植体已被广泛应用于牙缺失患者的口腔功能重建，但生理性牙周膜的缺失使得其在口颌复杂作用力及环境中比天然牙暴露出更多问题。已有研究证实种植体周牙周膜重建的可能性，但牙周膜成分复杂，纤维具有一定强度且排列有序，其形成依赖于精细的细胞内调控、细胞与细胞外基质的相互作用和萌出时承受的微应力。骨膜蛋白（Periostin）是一种细胞外基质蛋白，文献报道了其在牙发育、胶原生成、成骨及创伤愈合中的作用，并可在机械应力下维持牙周膜纤维系统和骨组织完整性，我们推测Periostin可能促进牙周膜干细胞在种植体表面的组织再生修复能力，并通过预实验发现其可促进牙周膜干细胞的成骨分化。本项目瞄准当前牙种植义齿缺乏牙周膜的关键问题，通过体外研究和动物模型，结合细胞学、分子生物学及干细胞膜片、钛表面处理等技术，观察并明确periostin对牙周膜干细胞在种植体周再生生理性牙周膜、组织维持的作用及其机制。

骨膜蛋白(Periostin, POSTN)在胶原生成及创伤愈合中起着重要作用，同时与成骨细胞的粘附、骨向分化密切相关；牙周膜干细胞(periodontal ligament stem cells, PDLSCs)作为种子细胞可定向分化形成牙周膜、牙槽骨、牙骨质。然而，POSTN在PDLSCs定向分化过程中的作用尚不清楚。本研究中，通过成功分离人PDLSCs，外源性POSTN刺激并构建POSTN低表达病毒，将病毒转染入干细胞，检测细胞骨、神经血管向分化能力。细胞增殖试验(cell counting kit-8,CCK8)、流式细胞术分析显示外源性POSTN刺激能显著促进PDLSCs的增殖；划痕实验表明外源性POSTN刺激能显著促进PDLSCs的迁移，而POSTN低表达组PDLSCs增殖、迁移能力较弱。茜素红染色结果显示，外源性POSTN刺激能显著促进PDLSCs的矿化，而POSTN低表达可降低PDLSCs的钙沉积。碱性磷酸酶(alkaline phosphatase, ALP)活性实验、蛋白免疫印迹法和实时逆转录聚合酶链反应进一步证实了成骨标记指标 (ALP, RUNX2, OSX, OCN, COL-1) 、成神经指标(β-tubulin Ⅲ, GFAP, NSE)在外源性POSTN刺激组细胞中明显上调。相反，POSTN低表达可以下调这些标记物的表达。此外，小管形成试验显示POSTN刺激能显著促进PDLSCs成血管分化。蛋白免疫印迹法表明炎症条件下，p-JNK在POSTN刺激的PDLSCs中被激活上调，但在POSTN低表达的PDLSCs中被抑制。POSTN可能通过调节JNK信号通路来调控PDLSCs的干细胞定向分化。本研究为确定牙周组织再生的干细胞靶点，为干细胞介导的牙周炎、种植体周围炎的治疗提供理论依据和实验基础，为POSTN对PDLSCs的功能改建及临床应用提供重要线索。