摘要
目的构建功能化组织工程骨是目前骨组织工程研究过渡到临床应用的重要方向。通过组织工程骨的神经化构建,在体内外实验的基础上探讨神经因素在骨组织工程中的作用。方法5～6月龄健康新西兰大白兔54只,雌雄不限,体重2～3kg,从骨髓中分离培养BMSCs;制备兔外周感觉神经匀浆和运动神经匀浆,按1∶10(V/V)加入LG-DMEM培养基,培养第2代BMSCs。对照组以LG-DMEM培养基培养。通过MTT实验、ALP染色和Ⅰ型胶原免疫细胞化学染色鉴定,观察感觉、运动神经匀浆在体外对BMSCs增殖和成骨分化的影响。将体外经成骨诱导的BMSCs复合β磷酸三钙(βtricalcium phosphate,β-TCP)制备组织工程骨,植入兔体内修复15mm股骨与骨膜缺损。将兔随机分为3组,每组18只,A组在β-TCP生物支架材料的侧槽中植入感觉神经束,B组植入运动神经束,C组为单纯组织工程骨;分别于术后4、8、12周通过X线片、骨密度检测和免疫组织化学染色观察神经化组织工程骨的成骨效应。结果MTT法检测示,各组吸光度(A)值随培养时间延长均逐渐增加,从6d开始,感觉神经组和运动神经组的A值均低于对照组(P<0.01);8d和10d时,感觉神经组A值低于运动神经组(P<0.05)。培养7d后,感觉神经组和运动神经组ALP染色阳性细胞数均明显少于对照组;培养14d后,感觉神经组和运动神经组的细胞未见Ⅰ型胶原阳性表达,对照组的细胞Ⅰ型胶原表达呈阳性。兔大段骨缺损修复区的术后X线片及Yang评分提示,随着时间延长,各组Yang评分逐步升高,8周后A组评分高于B、C组(P<0.01),B、C组间差异无统计学意义(P>0.05)。术后12周骨密度检测显示A组骨缺损修复良好,骨密度值高于B、C组(P<0.01),B、C组间差异无统计学意义(P>0.05)。术后12周组织工程骨成骨局部的免疫组织化学染色显示,S-100在A组表达量较高,B组表达量较少,C组偶见表达;降钙素基因相关肽在A组表达量高,B�
Objective Construction of viable tissue engineered bone is one of the most important research fields in the clinical application of bone tissue engineering,to investigate the function of nerve factors in bone tissue engineering by cell detection in vitro and construction of neurotization tissue engineered bone in vivo. Methods Fifty-four healthy New Zealand white rabbits,male or female,weighing 2-3 kg,were involved in this study. Bone marrow mesenchymal stem cells （BMSCs） from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates,using the LG-DMEM complete medium as control. The proliferation and osteogenic differentiation of the cells were observed and tested by the MTT assay,alkaline phosphatase （ALP） stain,and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in β tricalcium phosphate （β-TCP） biomaterial scaffold and cultured for 72 hours,then the β-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups （n=18）：sensory nerve bundle （group A） or motor nerve bundle （group B） were transplanted into the side groove of β-TCP scaffold,group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th,8th,and 12th weeks after operation.Bone mineral density （BMD） detection and S-100,calcitonin gene-related peptide （CGRP） immunohistochemistry stain were used at the 12th week to evaluate the effects of bone formation and discuss the mechanism. Results MTT assay indicated that the absorbance （A） value of each group increased with culture time. From the 6th day,the A values of both the sensory nerve and motor nerve homogenate groups were lower than that of the control group,showing significant difference （P〈 0.01）. On the 8th and 10th days,the A value of the sensory nerve homogenate group
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2010年第5期599-605,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
NSFC-广东省联合基金重点项目(u0732003)
国家重点基础研究发展计划(973)资助项目(2009CB930003)
国家自然科学基金资助项目(30600643)~~
关键词
组织工程骨
骨缺损
神经植入
BMSCS
兔
Tissue engineered bone Bone defect Neurotization Bone marrow mesenchymal stem cells Rabbit
