煅烧骨结合脱细胞真皮基质用于犬牙槽嵴的保持

Calcined bovine bone combined with acellular dermal matrix for maintaining the alveolar ridge in dog

背景:临床上常采用引导骨再生技术对骨缺损部位进行重建,即在骨缺损处植入支架材料,再采用生物屏障膜建立稳定的成骨环境,保证成骨细胞的增殖及血管的形成,取得了良好的效果。目的:观察煅烧骨结合脱细胞真皮基质用于拔牙窝填充的成骨效果。方法:采用高温煅烧法将牛松质骨制成煅烧骨,采用去表皮、脱细胞等方法将猪断层皮片制成脱细胞真皮基质,MTT法检测两种材料的细胞毒性。取9只比格犬,拔除下颚两侧第2,4前磨牙,拔牙窝内植入煅烧骨后用脱细胞真皮基质覆盖,分离颊舌侧牙龈并拉拢缝合,关闭拔牙创。术后1,3,6个月,采用锥形束CT、X射线片及组织学观察等方法评价成骨效果。实验由中国辐射防护研究院药物安全性评价中心伦理委员会审批,编号CIRP-IACUC-(R)2018081。结果与结论:(1)两种材料浸提液分别培养L929小鼠成纤维细胞24 h后,倒置显微镜下可见细胞形态良好,无空泡、死亡现象,MTT法结果显示细胞存活率均在85%以上,无明显的细胞毒性;(2)动物实验下颌骨锥形束CT检测显示,随着愈合时间延长,植入部位的相对灰度值逐渐增高,拔牙创内的成骨量增加;(3)动物实验X射线片显示,术后1个月,植入部位成像均匀、无明显成骨;术后3个月,植入部位有部分成骨;术后6个月,植入部位有明显成骨;(4)动物实验苏木精-伊红染色显示,术后1个月,拔牙窝内有大量新形成的骨小梁结构,骨小梁排列均匀,煅烧骨部分被吸收;术后3个月,见粗大骨小梁结构,伴有大量软骨,残留少量的煅烧骨;术后6个月,拔牙创接近愈合,形成成熟骨组织,可见骨单元结构,已观察不到煅烧骨;(5)结果表明,煅烧骨复合脱细胞真皮基质用于拔牙窝的填充具有良好的成骨效果。

BACKGROUND:In clinic,guided bone regeneration technology is often used to reconstruct the bone defect.That is,the scaffold material is implanted in the bone defect,and then the biological barrier membrane is used to establish a stable environment to ensure the proliferation of osteoblasts and the formation of blood vessels,which has achieved good results.OBJECTIVE:To observe the osteogenic effect of true bone ceramic combined with acellular dermal matrix in tooth socket filling.METHODS:Bovine cancellous bone was calcined to true bone ceramic.The acellular dermal matrix was prepared from split-thickness skin which the epidermis and cellular components have been extracted.Cytotoxicities of true bone ceramic and acellular dermal matrix were evaluated by MTT assay.The second and fourth premolars on both sides of the mandible were extracted in nine beagle dogs.The true bone ceramic was implanted into the socket and covered with acellular dermal matrix.The buccal and lingual gingivae were separated and sutured to close the extraction wound.At 1,3,and 6 months after surgery,cone beam CT,X-ray and histology were used to evaluate the new bone formation.The experiment was approved by Drug Safety Evaluation Center in China Institute for Radiation Protection(approval No.CIRP-IACUC-(R)2018081).RESULTS AND CONCLUSION:(1)L929 mouse fibroblasts were cultured with the two extracts for 24 hours.Under the inverted microscope,the cells were in good shape without vacuoles or death.MTT assay results showed that the cell survival rate was above 85%,without obvious cytotoxicity.(2)The results of cone beam CT showed that the relative gray value of the implant site increased gradually with the extension of healing time,and the amount of bone formation in tooth extraction wound increased.(3)The X-ray films showed that at 1 month after the operation,the imaging of the implanted site was uniform without obvious osteogenesis.At 3 months after the operation,there was partial osteogenesis.At 6 months after the operation,the implant site had obvious osteogenesis.(4)Hematoxylin-eosin staining showed that a large number of trabeculae were formed 1 month after operation,and the bone trabeculae was evenly arranged,and the true bone ceramic was partly absorbed.Coarse trabeculae structure with a large amount of cartilage and a small amount of calcined bone remained at 3 months after operation.The tooth extraction wound was nearly healed and mature bone tissue was formed,and the bone unit structure was visible;true bone ceramic could not be seen at 6 months after operation.(5)Results suggest that true bone ceramic combined with acellular dermal matrix show good bone formation for extraction socket filling.