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

本研究旨在研究几丁质和壳聚糖在大鼠牙槽骨内的组织化学降解过程。采用包埋后免疫金标法免疫组织化学检测溶菌酶。利用凝集素-胶体金电镜技术对其降解过程进行了超微结构观察。根据去乙酰化程度，专门制备了三组甲壳素：100%去乙酰化甲壳素（DDAC 100）；50% (ddac 50)；和0% （DDAC 0）。本免疫组化研究显示，DDAC 100组未检测到溶菌酶的表达。此外，电镜观察清楚地表明，植入壳聚糖的轮廓随着时间的推移而改变，并且在DDAC 50和100组的吞噬体中存在壳聚糖样碎片。这些发现有力地表明，骨组织中容易提供吞噬细胞，如多核细胞，并且吞噬作用比吞噬作用更有效。DDAC 100是骨外科和骨再生治疗的理想材料。另一项研究是评价壳聚糖单体（d -氨基葡萄糖盐酸盐）作为盖髓剂的适用性。通过体外和体内实验，研究壳单糖对大鼠创伤后细胞代谢和伤口愈合机制的影响。在成骨细胞培养3 d后，壳聚糖组碱性磷酸酶（ALP）活性显著升高。逆转录聚合酶链反应分析显示，壳聚糖诱导成骨细胞培养3 d后ALP mRNA表达增加，培养7 d后骨形态发生蛋白-2 mRNA表达增加。在添加壳聚糖单体的培养基中，成纤维细胞的炎性细胞因子白细胞介素-8的合成受到强烈抑制。在大鼠实验中评价组织病理学效应。1天后，与壳聚糖聚合物相比，炎症细胞浸润较弱。3天后，在壳聚糖单体附近观察到成纤维细胞显著增殖。炎性细胞浸润几乎完全消失。5 d后，成纤维细胞增殖进展，增生成纤维细胞周围出现成牙细胞。这些结果表明，本研究首次报道壳聚糖单体与壳聚糖聚合物相比，在创面愈合初期具有生物相容性稳定的作用。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