刺激骨化之齒槽黏膜細胞促進拔牙窩及顱骨缺損組織再生之探討

Abstract

研究目的 齒槽黏膜細胞 (AMCs) 屬於間質幹細胞，且高度表現幹細胞表面抗原。然而，AMCs促進牙周再生的有效性仍有爭議。本研究的目的是評估刺激骨化的AMCs (OAMCs) 促進拔牙窩和顱骨骨缺損組織再生之潛力。 研究材料與方法 AMCs由Sprague-Dawley大鼠中分離出來。透過流式細胞儀評估幹細胞表面抗原的表現和抗原性，並藉由油紅O (Oil Red O)、亞里西安藍(Alcian blue)和茜素紅S (Alizarin Red S)染色鑑定三系分化潛力。動物實驗驗證分為兩部分，第一部分透過拔牙窩（extraction socket model）探討AMCs對於拔牙傷口癒合及骨組織再生的影響。實驗方式為將大鼠雙側上顎第一大臼齒拔除，於拔牙窩中填入齒槽黏膜細胞球 (AMCp group) 及刺激骨化齒槽黏膜細胞球 (OAMCp group) 作為實驗組，對側拔牙窩則不放置任何材料作為控制組（control group）。動物於第7天和28天犧牲，透過實體觀察、微電腦斷層影像（micro-CT）以及免疫組織化學染色進行拔牙窩癒合及骨再生的分析。 第二部分則是透過顱骨骨缺損（calvarial osseous defect model）探討AMCs對於骨再生效果的影響。實驗方式為在大鼠顱骨處利用環鑽工具製備直徑5毫米的圓型缺損，除了未放置任何材料的控制組（control group），實驗組於骨缺損中填入冷凍乾燥骨 (FDBA) 作為支架，分別加入水膠 (s-Hydrogel group)、齒槽黏膜細胞球 (s-AMCp group) 及刺激骨化齒槽黏膜細胞球 (s-OAMCp group)。動物於第7天和28天犧牲，透過微電腦斷層影像（micro-CT）以及免疫組織化學染色進行顱骨骨缺損之骨再生分析。 結果 AMCs高度表達幹細胞表面抗原、具有低抗原性，並且有三系分化能力。在拔牙窩模型中，與control group相比，AMCp group和OAMCp group傷口癒合和上皮角質化較為快速。Micro-CT影像分析顯示AMCp group和OAMCp group在第28天會觀察到比control group更多的新骨形成。與control group相比，AMCp group與OAMCp group的ERD數值顯著較低 (p < 0.01)。在第7天，K-10在AMCp group和OAMCp group的表現較control group明顯。OAMCp group的增殖細胞百分比在第7天跟28天皆顯著高於control group（p < 0.05)。 在顱骨骨缺損模型中，micro-CT影像分析顯示s-AMCp group和s-OAMCp group在第28天可觀察到有更多的礦化組織百分比 (p < 0.05)。相較於control group、s-Hydrogel group及s-AMCp group，在第7天可以觀察到BSP在s-OAMCp group的骨缺損邊緣及骨粉周圍就有明顯表現。同樣在第7天的組織切片，s-OAMCp group的增殖細胞百分比顯著比對照組高 (p < 0.01)。 結論 本實驗的結果顯示AMCs高度表現幹細胞表面抗原、具低抗原性，且有三系分化能力。AMCs能加速拔牙傷口癒合，OAMCs可以促進顱骨缺損再生，可推論OAMCs對於牙周再生有正向幫助。

Objective Alveolar mucosal cells (AMCs) are mostly of the mesenchymal lineage and highly express stem cell markers. However, the effectiveness of AMCs in promoting periodontal regeneration is still debated. The aim of this study was to evaluate the potential of osteogenically stimulated AMCs (OAMCs) to promote extraction socket and calvarial bony defect regeneration. Materials and Methods AMCs were isolated from Sprague-Dawley rats. The expression of stem cell surface markers and antigenicity were assessed using flow cytometry, and trilineage differentiation potential was identified via Oil Red O, Alcian Blue, and Alizarin Red staining. Extraction sockets and calvarial osseous defects were surgically created in Sprague-Dawley rats, and the sockets/defects were unfilled, filled with AMCs, or filled with OAMCs. Gross observation, micro computed tomography (CT) imaging, histologic evaluation, and immunohistochemistry for proliferating cells and expression of cytokeratin, were conducted in the extraction socket wounds at 7 and 28 days. Micro-CT imaging, histologic evaluation, and immunohistochemistry for proliferating cells and expression of bone sialoprotein (BSP) were conducted in the osseous defects at 7 and 28 days. Results AMCs highly expressed stem cell surface markers with weak antigenicity and were capable of adipogenic, chondrogenic, and osteogenic differentiations. In the extraction socket model, wound closure, socket fill, and keratinization were significantly accelerated in those with AMCs and OAMCs relative to the unfilled wounds. The micro-CT images revealed more new bone formation in the AMC spheroid (AMCp) and OAMC spheroid (OAMCp) groups than in the control group on Day 28. The edentulous ridge discrepancy of the AMCp and OAMCp groups was significantly lower than that of the control group (p < 0.01). K-10 was only slightly expressed in the epithelium of the extraction wound in the control group but was widely expressed in those of the AMCp and OAMCp groups. The percentage of proliferating cells was significantly higher in the OAMCp group than in the control group on Days 7 and 28 (p < 0.05). In the calvarial osseous defect model, the micro-CT images revealed a greater mean bone volume/defect volume ratio in the s-AMCp and s-OAMCp groups than in the control group (p < 0.05). On Day 7, BSP was lightly deposited at the defect peripheries in the control, s-Hydrogel, and s-AMCp groups, and was widely spread around the borders and graft granules of the s-OAMCp group. The percentage of proliferating cells was higher in the s-OAMCp group than in the control group on Day 7 (p < 0.01). Conclusion AMCs highly expressed stem cell surface markers with weak antigenicity and were capable of trilineage differentiation. AMCs accelerated extraction socket wound healing, and OAMCs facilitated calvarial bony defect regeneration, supporting the notion that OAMCs promote periodontal regeneration.