Tissue engineered periodontal regeneration using human autologous derived cells

本项目的目的是利用组织工程技术来修复牙周病引起的牙周退缩和骨下缺损。要达到这一要求，需要细胞、生长因子和支架三个重要因素。特别是，在细胞供应方面，从一个小标本培养到适当大小的片材在临床上是可能的。人培养的牙龈上皮(HCGE)片或人的培养的牙龈成纤维细胞(HCGF)片是用Green和Hata或Kuroyanagi的改进方法制备的。临床应用HCGE片剂治疗脱屑性牙周炎，应用HCGF片剂治疗牙龈退缩。此外，还进行了人培养骨膜(HCP)的制备过程。术后12个月，HCP片剂、富含血小板血浆(PRP)和羟基磷灰石(HA)颗粒的联合治疗效果明显优于PRP加HA颗粒。一个可能促成这些良好临床结果的因素是，在HCP片层中存在成骨细胞，这提供了更大的再生潜力。为了获得上述组织工程实验的科学依据，我们进行了一些体外实验：肝细胞生长因子对成骨细胞或牙周膜(PDL)细胞的影响，细胞外ATP和ATPrS对PDL细胞的作用机制。作为下一阶段，我们开始挑战培养骨。对于培养骨，经酸蚀表面处理后，已建立的人成骨细胞很容易渗入HA块的孔底。此外，我们还尝试了PDL细胞在聚乳酸针织片材表面的三维培养。在聚乳酸针织片材中加入阿特罗-胶原凝胶，与单独使用聚乳酸针织品培养相比，更容易培养出更多的细胞。

The aim of this project is In regenerate gingival recession and infrabony defect caused by periodontal disease using tissue engineered technique.To achieve this requirement, three important factors such as cells, growth factors and scaffold are known to be needed. Especially, regarding cell supply, it was possible to practice in clinical situation by being cultured from a small specimen to an appropriate sire of sheet The human cultured gingival epithelial (HCGE) sheet or human cultured gingival fibroblast (HCGF) sheet was prepared by modified methods of those developed by Green and Hata, or Kuroyanagi. The HCGE sheet was applied clinically to treat an desquamative gingivitis, and the HCGF sheet was applied to treat gingival recession. Moreover, the human cultured periosteum (HCP) sheet preparation procedure was performed. Treatment with a combination of HCP sheets, platelet-rich plasma (PRP) and hydroxyapatite (HA) granules, when compared to PRP with HA granules, led to a significantly more favorable clinical improvement after 12-months post-surgery. A factor likely contributing to these favorable clinical results would be the presence of osteogenic cells in the HCP sheets which provided greater regeneration potential. To obtain scientific evidences of these tissue-engineered trials described above, we performed some in vitro experiments: the effect of hepatocyte growth factor on osteoblasts or periodontal ligament (PDL) cells, the mechanism of extracellular ATP and ATPrS on PDL cells. As a next stage, we began to challenge to develop cultured bone. Regarding cultured bone, established human osteoblasts were easily infiltrated into the bottom of pore of HA block after treatment of surface condition by acid-etching. In addition, we tried to 3-D culture of PDL cells on the surface of polylactic-acid (PLA) knit sheet. By adding atelo-collagen gel to PLA knit sheet, many cells were cultured easily compared with culture on only PLA knit alone.