屏障膜联合自体骨髓基质细胞修复牙槽骨缺损的实验研究

The study of barrier membrane combined with autogenous bone marrow stromal cells on alveolar bone defect in dogs

目的初步探讨屏障膜与骨髓基质细胞(marrow stromal cells,MSCs)结合后在牙槽骨缺损修复领域的应用潜能。方法①细胞培养:采用犬来源的MSCs体外扩增培养,观察检测非诱导条件下MSCs的生长变化和成骨分化。②动物实验:拔除3只雄性杂种犬的双侧上下颌第一、二前磨牙,保留唇舌侧牙槽嵴,制备一近远中向15mm×5mm,冠根向8mm的骨内缺损,随机分3组予以下处理:a.空白对照组:直接缝合牙龈;b.单纯膜组:骨缺损区上覆盖Bio-Gide膜后缝合牙龈;c.膜+MSCs:缺损区植入自身MSCs-胶原膜复合物,细胞朝向骨面,覆盖Bio-Gide膜后缝合牙龈,分别于术后4,8,12周随机取材,进行大体、X线和组织学检测。结果形态学观察表明,MSCs贴壁细胞呈集落生长,有成纤维细胞样外观,未加入成骨诱导剂,细胞形态发生变化,钙沉积出现,碱性磷酸酶(Alkalinephosphatase,ALP)表达。3代内扩增的MSCs有成骨活性,原代细胞成骨活性优于传代后细胞。动物实验表明,两盖膜组较空白对照组在8周内有明显的骨形成,但胶原膜加用MSCs在促进骨形成上未见明显区别。结论MSCs在体外培养能大量扩增,具有自然向成骨细胞分化的能力。使用Bio-Gide胶原膜进行引导骨再生术(GBR)可促进成骨,并且与GBR联用MSCs差异不明显,提示两者联合应用还有待进一步研究。

Objective To evaluate the bone formation of bioresorbable eollagen membrane used alone or in combination with MSCs in dog alveolar bone defeets. Methods （1） The MSCs obtained from mongrel dogs were eultured and subeultured. The proliferation and osteogenie differentiation of MSCs were observed. （2） Three male mongrel dogs were used. The first and seeond premolars were extraeted from the bilateral maxillary and mandibular bone and four alveolar intrabone defeets（ 8 mm in height, 5 mmin width, 15 mm in length） were ereated. The surgieally- ereated defeets were randomly assigned to one of the treatments： （A） control （nothing was used）, （B） GBR（only Bio- Gide）, （C） GBR＋ MSCs （ Bio - Gide ＋ MSCs）. The dogs were saefifieed at 4, 8, and 16 weeks after surgery. The maeroseopie,radiographic and histologieal proeessing were performed. Results （1） The euhured MSCs eomprised a single phenotypie population and displayed a fibroblast - like morphology. Without induetion eondition, eells ehanged from spindle - shape to euboidal and polygonal in eell morphology. With 3 passages process of extensive subeultivation, alkaline phosphatase （ALP） expression and deposition of ealeium salt were observed in the eulture. （2） The amount of newly formed bone was signifieandy greater in the groups using the membrane than that in the control group within 8 weeks. However, there were no significant differences between the use of the collagen barrier membrane alone or in combination with MSCs in enhancing new bone formation. Conclusion The proliferation and osteogenie differentiation of the MSCs were observed. The cell can be used as an ideal cell in therapy of bone defect. The results of this study indicate that GBR treatment with collagen membranes may significantly enhance bone regeneration within 8 weeks. The influence of GBR technique in combination with MSCs in accelerating the repair of alveolar bone defects is not clear.