The study on organic bioactivative materials for clinical application in the tissue regeneration medicine

在动物实验中，利用凝集素（WGA-gold）和溶菌酶作为免疫细胞化学标记物，研究了几丁质/壳聚糖填充骨缺损的生物降解过程。材料与方法选用6周龄雄性Wistar大鼠60只。甲壳素及其衍生物分为三组，每组20个样品，以下分别称其脱乙酰化衍生物为DAC 0、DAC 50、DAC 100。手术入路是通过下颌骨下缘皮肤切口。骨膜瓣抬高后，在颏孔与下颌骨下缘之间的骨表面形成一个进入骨髓的空腔。在解剖显微镜下，在每只大鼠下颌骨外侧外斜隆突下方各制备4个圆柱形空腔。将棉花状三种不同程度的脱乙酰甲壳素分别植入3个不封口的空腔，分别命名为DAC（脱乙酰甲壳素度）0、50和100组。术后1、2、3、4周仔细解剖植入的壳聚糖及周围骨，固定后用LR金包埋。未脱矿的各组标本用玻璃刀切开进行形态学研究。半薄切片（约1 μm）用甲苯胺蓝染色，光镜下用兔多克隆抗人溶菌酶进行免疫细胞化学研究。我们还通过透射电子显微镜检查了薄切片上几丁质残体超微结构定位的高分辨率视图。结果与讨论植入DAC 0后，术后2周内炎症细胞浸润消失，壳聚糖纤维周围溶菌酶积累持续4周以上。然而，它们的外观程度随着时间的推移而降低。DAC 50组，1周内早期炎症消失，2周未发现溶菌酶积累。DAC 100组，1周未见早期炎症和溶菌酶积累。说明壳聚糖去乙酰化程度越高，其生物降解和骨修复的速度越快。本研究表明，高度去乙酰化的壳聚糖在拔牙、根尖切除和牙周手术等临床治疗中具有快速骨愈合的潜力。少

IntroductionThis reseach focus on the biodegradation process of the chitin/chitosan filled in the bone defect at the animal study by the useage of lectin (WGA-gold) and the lysozyme as the immuocytochemical marker.Materials and methodsSixty, 6-weeks-old male, Wistar rats were used for this experiment. Chitin and derivatives were divided into three groups of twenty specimens each, hereafter we will call the deacetylated derivatives DAC 0,DAC 50,DAC 100,respectively. The operation approach was made through a skin incision along the inferior border of the mandible. After periosteal flap was elevated, a cavity was prepared into the bone marrow from the bone surface between the mental foramen and the inferior border of mandible. Under a dissecting microscope, four cylindrical cavities were prepared below the external oblique protuberance on each lateral sides of the mandible for each rat. The cotton-like three kinds of different degree of deacetylated chitin was implanted in the each three … More cavities without sealing, respectively, designated DAC(degree of deacetylated chitin) 0,50 and 100 group. At 1,2,3 and 4 weeks postoperatively, the implanted chitosan together with surrounding bone were carefully dissected At the post fixation, the blocks were embedded in LR gold. Undemineralized each group specimens were cut with glass knives for the morphological study. Semithin sections (about 1 μm) were stained with toluidine blue for light microscopy and for the immunocytochemical study we used rabbit polyclonal anti-human lysozyme. We also examined a high-resolution view of ultrastructual localization of chitin remnant on thin sections by the transmission electron microscope.Results and DisccusionIn case of implanting DAC 0,inflammatory cells infiltration was disappeared within 2 weeks postoperatively and the accumulation of lysozyme surrounding chitosan fibers was continued more than 4 weeks. However their degree of the appearance was reduced with time. In case of DAC 50,the early stage inflammation was disappeared within 1 week and the accumulation of lysozyme was not recognized by 2 weeks. In case of DAC 100,the early stage inflammation and the accumulation of lysozyme were not observed by 1 week. These findings suggest that the higher the degree of deacetylation of chitosan, the more rapid the biodegradation and the bone repair. The present study demonstrates that the highly deacetylated chitosan is possible to apply in clinical dentistry such as teeth extraction, apicectomy and periodontal surgery for the rapid bone healing. Less