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软骨脱细胞基质支架材料的软骨组织工程实验研究
引用本文:张晨,景士兵,杨琨,徐燕莉,聂军,王燕妮,文晓泉,高景恒.软骨脱细胞基质支架材料的软骨组织工程实验研究[J].中国修复重建外科杂志,2008,22(7):846-850.
作者姓名:张晨  景士兵  杨琨  徐燕莉  聂军  王燕妮  文晓泉  高景恒
作者单位:1. 大连大学附属新华医院整形外科,辽宁大连,116021
2. 辽宁省人民医院病理科
3. 辽宁省人民医院中心实验室
4. 辽宁省人民医院整形外科
摘    要:目的应用软骨脱细胞基质作为支架材料,按照组织工程的原理再生软骨,为修复软骨缺损探索新的途径。方法取新西兰大耳白兔1只,体重2.4kg,按改良Courtman法对兔耳软骨行脱细胞处理后用于实验。选用纯种6月龄新西兰大耳白兔18只,雌雄不限,体重2.4~2.6kg。每只耳形成2处1cm×1cm软骨缺损,按修复方式不同随机分为3组,每组24处缺损。A组,软骨脱细胞基质加软骨膜;B组,软骨脱细胞基质;C组软骨膜,作为对照。术后每日观察兔耳修复区的大体变化,并分别于4、12周每组处死3只动物,于修复区切取标本,行HE染色、藏红花红一奥尔新蓝染色、II型胶原基因探针原位杂交实验。结果术后4周内A、B组大体形态无明显改变;术后12周可见修复区轻度增厚,触摸时质地较正常软骨稍硬。C组术后2周,2只2处修复区形成痂皮,术后5周痂皮脱落形成穿孔。术后4周,A、B组HE染色可见软骨脱细胞基质周边有轻度的炎性细胞浸润,以淋巴细胞为主,未形成包囊,藏红花红.奥尔新蓝染色均阴性;C组软骨缺损处的软骨膜塌陷,无细胞增殖现象;各组修复区均未见II型胶原基因探针原位杂交显色。术后12周,A组HE染色示部分软骨脱细胞基质内有新生细胞,细胞排列不规律,ECM嗜碱性增强,藏红花红-奥尔新蓝染色呈阳性,II型胶原基因原位杂交显色示新生细胞内均有大片棕黄色阳性染色区域;B组HE染色示软骨脱细胞基质无吸收现象,周边无包囊,炎性细胞消失,藏红花红一奥尔新蓝染色呈阴性,未见II型胶原基因原位杂交显色;C组HE染色示近软骨断端处可见部分形成新生组织,藏红花红一奥尔新蓝染色呈阳性,II型胶原基因原位杂交显色可见棕黄色阳性染色细胞。结论脱细胞软骨可诱导软骨膜细胞向其中生长而重建软骨,软骨膜和脱细胞软骨复合移植是软骨组织工程的另一种选择。

关 键 词:组织工程软骨  耳软骨脱细胞基质  软骨膜    软骨组织工程  脱细胞基质  支架材料  工程实验  研究  MODEL  RABBIT  SCAFFOLD  MATRIX  CARTILAGE  ACELLULAR  选择  复合移植  重建  生长  骨膜细胞  分形  骨断端  炎性细胞  吸收现象

CARTILAGE TISSUE ENGINEERING WITH ACELLULAR CARTILAGE MATRIX AS SCAFFOLD IN RABBIT MODEL
ZHANG Chen,JING Shibing,YANG Kun,XU Yanli,NIE Jun,WANG Yanni,WEN Xiaoquan,GAO Jingheng.CARTILAGE TISSUE ENGINEERING WITH ACELLULAR CARTILAGE MATRIX AS SCAFFOLD IN RABBIT MODEL[J].Chinese Journal of Reparative and Reconstructive Surgery,2008,22(7):846-850.
Authors:ZHANG Chen  JING Shibing  YANG Kun  XU Yanli  NIE Jun  WANG Yanni  WEN Xiaoquan  GAO Jingheng
Institution:Division of Plastic Surgery, Xinhua Hospital of Dalian University, Dalian Liaoning, 116021, PR China. neilzhang@sina.com
Abstract:OBJECTIVE: To study how to repair the cartilage defect according to the principles of tissue engineering with acellular cartilage matrix as scaffold material. METHODS: The ear cartilage was obtained from a New Zealand white rabbit (weighing 2.4 kg )and then treated by a modified Courtman's four-step method to produce the acellular cartilage matrix. Eighteen New Zealand white rabbits (aged 6 months, weighing 2.4-2.6 kg) with no sex limit were divided into three groups. For every rabbit, two pieces of ear cartilage measured 1 cm x 1 cm were excised in each ear. Defects were repaired as follows: group A with the combined graft of acellular cartilage matrix and perichondium, group B with acellular cartilage matrix and group C with perichondium. Three animals in each group were killed 4 and 12 weeks postoperatively, respectively. Tissue samples obtained were analyzed with gross observation, hematoxylin-eosin stain, Safranine O-alcian blue stain and type II collagen messenger RNA in situ hybridization respectively. RESULTS: In gross observation, the repaired sites in groups A and B were not change meaningfully in their shape 4 weeks postoperatively; but they felt a bit of thicker and harder 12 weeks postoperatively. In group C two repaired sites formed scabs at 2 weeks and perforated at 5 weeks. In histological observation, there was a slight inflammatory reaction surrounding the acellular cartilage matrix 4 weeks after it was implanted in groups A and B. The inflammatory cells were mainly lymphocytes. The perichondrium graft in group C was collapsed in the defects in HE stain. The defect sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization in all groups. At 12 weeks cells were found in the acellular matrix which arranged in irregular manner in group A in HE stain and was positive for Safranine O-alcian blue stain and type II collagen mRNA in site hybridization. In groups B and C, no new cell was found in HE stain and the repaired sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization. CONCLUSION: Acellular cartilage matrix is a potential scaffold material to the cartilage tissue engineering. The combined graft of acellular cartilage matrix and perichondium can be an alterative method for the repair of the wounded cartilage.
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