Creation and clinical application of the three-dimensional cultured cell transplantation for hard tissue regeneration

本研究旨在用组织化学方法研究甲壳素和壳聚糖在大鼠牙槽骨中的生物降解过程。包埋后免疫金标记法进行溶菌酶免疫组织化学检测。用电子显微镜结合凝集素-胶体金技术对降解过程进行了超微结构观察。根据甲壳素脱乙酰度的不同，制备了三组甲壳质：100%脱乙酰甲壳素(DDAC100)、50%脱乙酰甲壳素(DDAC50)和0%脱乙酰甲壳素(DDAC0)。免疫组织化学研究表明，DDAC100组未检测到溶菌酶的表达。此外，电子显微镜清楚地显示了植入壳聚糖的轮廓随时间的变化，并且在DDAC50和100组的吞噬小体中存在壳聚糖样碎片。这些发现有力地表明，吞噬细胞，如多核细胞，很容易在骨组织中供应，吞噬作用比EN…更有效。更多的酶消化甲壳素和壳聚糖在骨组织中的生物降解性。DDAC 100是一种适合骨外科和骨再生治疗的生物材料。另一项研究是评价壳聚糖单体(D-氨基葡萄糖盐酸盐)作为盖髓药物的适用性。采用体外实验和体内实验相结合的方法，研究了壳聚糖对创面细胞代谢和创面愈合机制的影响。成骨细胞培养3d后，壳聚糖组碱性磷酸酶(ALP)活性显著升高。逆转录聚合酶链式反应分析显示，壳聚糖诱导成骨细胞培养3d后碱性磷酸酶基因表达增加，7d后骨形态发生蛋白-2基因表达增加。在添加壳聚糖单体的培养液中，成纤维细胞中炎性细胞因子IL-8的合成受到强烈抑制。在大鼠实验中评价其组织病理学效应。1天后，与壳聚糖聚合物组相比，炎性细胞浸润较轻。3天后，所用壳聚糖单体附近可见成纤维细胞显著增殖。炎性细胞浸润几乎完全消失。5d后成纤维细胞进一步增殖，增殖的成纤维细胞周边可见一些成牙本质细胞。这些结果表明，与壳聚糖聚合物的应用相比，本研究首次报道了壳聚糖单体作为一种生物相容性稳定的药物，即使在伤口愈合的早期阶段也是如此。较少

The present study was designed to investigate histochemically the biodegradation processes of chitin and chitosan implanted in rat alveolar bone. Lysozyme was immunohistochemically detected using postembedding immunogold labeling. The degradation process was ultrastructurally observed using the lectin-colloidal gold technique with electron microscopy. Three groups of chitin were specially prepared according to their degree of deacetylation : 100% deacetylated chitin (DDAC 100) ; 50% (DDAC 50) ; and 0% (DDAC 0). The present immunohistochemical study indicated that lysozyme expression was not detected in the DDAC 100 group. Furthermore, electron microscopy clearly demonstrated that the contour of implanted chitosan changed over time, and that chitosan-like fragments were present in the phagosomes in the DDAC 50 and 100 groups. These findings strongly suggest that phagocytes, such as multinuclear cells, are easily supplied in bone tissue and that the phagocytosis is more effective than en … More zymatic digestion for chitin and chitosan biodegradation in bone tissue. DDAC 100 should be a suitable biomaterial for bone surgery and bone regeneration therapy.The other study was undertaken to evaluate the applicability of chitosan monomer (D-glucosamine hydrochloride) as a pulp capping medicament. Both in vitro and in vivo experiments were carried out to study the cell metabolism and wound healing mechanisms following the application of chitomonosaccharide. After 3 days of osteoblast culture, alkaline phosphatase (ALP) activity significantly increased in the chitosan group. Reverse transcription polymerase chain reaction analysis revealed that chitosan induced an increase in the expression of ALP mRNA after 3 days and bone morphogenetic protein-2 mRNA after 7 days of osteoblast incubation. Inflammatory cytokine, interleukin (IL)-8, synthesis in fibroblasts was strongly suppressed in the medium supplemented with chitosan monomer. Histopathological effects were evaluated in rat experiments. After 1 day, inflammatory cell infiltrations were observed to be weak when compared with the application of chitosan polymer. After 3 days, a remarkable proliferation of fibroblasts was seen near the applied chitosan monomer. The inflammatory cell infiltration had almost completely disappeared. After 5 days, the fibroblastic proliferation progressed, and some odontoblastic cells appeared at the periphery of the proliferated fibroblasts. These findings indicate that the present study is the first report that chitosan monomer acts as a biocompatibly stable medicament even at the initial stage of wound healing in comparison with the application of chitosan polymer. Less