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

本研究以凝集素(WGA-Gold)和溶菌酶为免疫细胞化学标记物，研究了甲壳素/壳聚糖充填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。甲壳素及其衍生物被分成三组，每组20个，此后我们将分别称为脱乙酰化的衍生物DAC0，DAC50，DAC100。手术入路为下颌下缘皮肤切开入路。骨膜瓣抬高后，从颧孔至下颌骨下缘之间的骨面向骨髓内预备一个空洞。在解剖显微镜下，每只大鼠双侧下颌骨外斜突下方制作4个圆柱形空洞。将三种不同脱乙酰度的棉状甲壳素分别植入三种…中空洞多不封闭，分别命名为DAC(脱乙酰度甲壳质)0、50、100组。术后1、2、3、4周，在固定后仔细解剖植入的壳聚糖及周围骨，块内埋入LR金。未脱钙的各组标本用玻璃刀切割，进行形态观察。半薄切片(约1μm)用甲苯胺蓝染色进行光镜观察，免疫细胞化学研究用兔多克隆抗人溶菌酶。结果与讨论植入DAC0后，术后2周内炎性细胞浸润消失，溶菌酶在壳聚糖纤维周围积聚超过4周。然而，它们的出现程度随着时间的推移而降低。DAC50组1周内早期炎症消失，2周未见溶菌酶积聚。DAC-100组1周后未见早期炎症和溶菌酶积聚。这些结果表明，壳聚糖的脱乙酰度越高，其生物降解和骨修复越快。本研究表明，高度脱乙酰化壳聚糖具有骨愈合快的特点，可应用于拔牙、根尖切除、牙周手术等临床牙科领域。较少

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