Fundamental research of application of self periodontium derived cells spheroid to guided tissue regeneration

牙周治疗的最终目的是使受损的牙周组织再生，恢复原来的形态和功能。据认为，新骨、新骨质和新结缔组织的再粘连是被破坏的组织再生所必需的。我们以前报道过这种球体是由牙周韧带成纤维细胞产生的，可能用于牙周重建治疗。由于球体具有球形的三维结构，含有具有细胞增殖活性的细胞，并产生细胞外基质蛋白。这次，我们使用含有胎牛血清培养基（D-MEM）的人牙槽骨源性细胞（HABC）产生球体。我们使用针对i型胶原、骨桥蛋白（OP）、骨钙素（OC）和Ki-67抗体的单克隆抗体对球状体进行了免疫组织化学研究。另一种方法是在无血清培养基中建立重组人血小板衍生生长因子（rhPDGF）和重组人b-成纤维细胞生长因子（rhb-FGF）的培养方法，引入球状体对牙周组织的再生治疗。Ki-67的阳性反应在第1天和第3天出现在整个球体中，而在第7天和第21天只出现在表层。在第1天至第21天观察到i型胶原蛋白，在第1天至第7天观察到整个球体有碱性磷酸酶。在21^<st>天，HABC可以在球体内表面产生OC和OP。在无血清培养基中添加100 ng/ml rhb-FGF的培养中，第5天的生长速度最高，为D-MEM培养的42.0%。HABC在球状环境下表达i型胶原、OC和OP，因此球状体可能用于牙周重建治疗。

The ultimate goal of periodontal therapy is the regeneration of destroyed periodontal tissues, and is in the original form and recovery of a function. It is thought that the re-adhesion by the new bone, new cementum, and new connective tissue is required for the destroyed rebirth of an organization. We previously reported that the spheroid was produced with periodontal ligament fibroblasts and might be used for periodontal reconstructive therapy. Because spheroid has spherical 3-dimensional structure and that contained the cells which have cell proliferation activity, and produced extracellular matrix proteins.This time, we produced spheroid using the human alveolar bone derived cell(HABC) with containing fetal calf serum medium(D-MEM). We performed an immunohistochemical study for spheroids using the monoclonal antibody against collagen type-I, osteopontin(OP), osteocalcin(OC) and Ki-67 antibodies. Another approach is to establish of the culture method with growth factor which were a recombinant human platelet derived growth factor(rhPDGF) and a recombinant human b-fibroblast growth factor(rhb-FGF), in the serum free medium to introducing the periodontal tissue regeneration therapy with the spheroids.The positive reaction of Ki-67 was observed in the whole of spheroid on the first day and the third day, but observed only in the surface layer on the seventh day and the 21^<st> day. Collagen type-I was observed on the fist day to on the 21^<st> day and an alkaline phosphatase was observed in the whole spheroid on the first day to 7^<th> day. HABC could produce OC and OP at inside surface of the spheroid on the 21^<st> day. The culture with serum free medium contained with 100 ng/ml rhb-FGF appeared the highest growth rate was 42.0 % of the culture with D-MEM at the 5 day. HABC expressed Collagen type-I, OC and OP in the spheroid environment, so spheroids may be capable of being applied to periodontal reconstructive therapy.