北京大学医学部机构知识库
Advanced  
IR@PKUHSC  > 北京大学第三临床医学院  > 学位论文
学科主题: 运动医学
题名:
骨形态发生蛋白4基因修饰的脂肪干细胞复合藻酸钙凝胶支架对骨软骨镶嵌成形术修复巨大软骨损伤的作用研究
作者: 陈临新
答辩日期: 2015-12-31
导师: 敖英芳
专业: 运动医学
授予单位: 北京大学
授予地点: 北京大学第三临床医学院
学位: 博士
关键词: 软骨损伤 ; 骨形态发生蛋白4,脂肪干细胞 ; 藻酸钙凝胶 ; 自体骨软骨镶嵌移植术
其他题名: cartilage defect,morphogenetic protein 4;dipose-derived stem cells;Mosaicplasty;CaAlg hydrogel
分类号: R681.3
摘要:

大面积的软骨损伤的修复是一个难题,骨软骨镶嵌成形术(Mosaicplasty)是解决这一问题的方法之一,但移植软骨与周围整合不良,存在“死区”。为解决这一问题,本课题利用可注射式藻酸钙水凝胶填充Mosaicplasty后遗留在移植软骨之间的空隙,在藻酸钙水凝胶中复合骨形态发生蛋白4(bone morphogenetic protein4,BMP4)修饰的脂肪干细胞(adipose-derived stem cells,ADSCs),以促进移植软骨与周围软骨的整合,减少“死区”的发生。

[方法]

体外试验:分离、培养ADSCs,通过检测多向诱导分化能力及流式细胞术检测干细胞标记物,完成ADSCs的干细胞特性鉴定。BMP4-腺病毒载体转染ADSCs48小时后,利用流式细胞仪检测转染效率;同时免疫荧光染色观察BMP4的表达情况。酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)测定细胞培养液及裂解液中的BMP4蛋白和I型胶原、II型胶原蛋白以及Sox9,ALP的含量。

动物试验:以小型猪为研究对象,在股骨内髁负重区做一个直径8.5mm,深5mm缺损;在股骨滑车边缘非功能区分别取3个直径3.5mm,高5mm骨软骨柱,用于Mosaicplasty技术填充负重区的大部分缺损面积。藻酸钙水凝胶注射式填充Mosaicplasty技术后残留的软骨“死区"。注射的藻酸钙凝胶内含有转染BMP4-腺病毒后24小时 的ADSCs。24只小型猪随机平均分为3组: 组Ⅰ为单纯Mosaicplasty组,组Ⅱ为Mosaicplasty+藻酸钙凝胶组,组Ⅲ为Mosaicplasty+藻酸钙凝胶+ BMP4-ADSCs组。分别在术后12周,24周取标本进行核磁共振、组织学大体观察、组织学染色(HE染色、甲苯胺蓝)、免疫组化(Ⅱ型胶原)染色、组织学评分以及扫描电镜观察,生物力学检测。定量结果用均数±标准差表示,统计学分析使用SPSS 软件,P<0.05为有统计学意义。

[结果]

体外实验:分离培养ADSCs,取第三代进行多向诱导分化及干细胞表面标记鉴定。在特定诱导条件下,ADSCs 可分化为成脂细胞、成软骨、成骨细胞。同时,流式细胞术检测ADSCs间充质来源的表面标志分子CD90和CD44呈阳性率分别为99.85%和99.22%,而血液来源的表面标志分子CD34阳性率仅为11.17%,证实ADSCs具有干细胞特性。BMP4基因重组的腺病毒载体对ADSCs 的转染效率为98.86±0.74%。转染后48h,通过免疫荧光检测到ADSCs中 BMP4蛋白的表达。ELISA检测:转染BMP4的ADSCs裂解液中BMP4蛋白和I型胶原、II型胶原蛋白、Sox9、ALP的含量较单纯ADSCs培养液中的含量明显增加。

动物实验:核磁共振及扫描电子显微镜检测均发现组Ⅰ与组Ⅱ移植软骨与周围未能愈合,存在间隙,只有组Ⅲ移植软骨与周围愈合。大体观察及组织学切片表明组Ⅰ移植软骨与正常组织间裂隙明显,修复组织少,且为纤维瘢痕组织;组Ⅱ移植软骨与正常软骨间有部分新生修复组织,但仍存在裂隙,修复组织并非透明软骨组织;组Ⅲ移植软骨与周围软骨间完全融合,表面连续完整,新生软骨组织甲苯胺蓝及Masson染色阳性,Ⅱ型胶原免疫组化染色阳性。12周时三组的组织学评分分别为5.21±1.60,8.83±3.12,16.20±2.63。24周时三组的组织学评分分别为:7.24±1.32,12.15±4.56,21.42 ±1.35。组Ⅲ组织学评分明显高于其余两组,接近正常软骨。24周标本生物力学检查结果:三组的接触刚度(N/M)分别为2.88±0.97,9.02±4.33,29.87±7.59,弹性模量(Mpa)分别为0.14±0.05,0.45±0.21,1.44±0.50 ,硬度(Kpa)分别为5.36±3.22,11.38±6.92,22.51±7.29。组Ⅲ的接触刚度、弹性模量与正常软骨接近,硬度较正常软骨差,但三个参数均明显好于组Ⅰ及组Ⅱ的测量值。

[结论]

BMP4修饰的ADSCs结合藻酸盐水凝胶支架弥补了Mosaicplasty技术的不足,在软骨之间的裂隙生成类透明软骨,能够促使移植软骨与周围软骨完全整合,减少“死区”的出现。

英文摘要:

[Background]

Cartilage repair still presented a challenge to clinicians and researchers alike. Mosaicplasty was a feasible method to solve this problem. However, "dead zone" existed in the implanted cartilage and the surrounding native tissue. In order to solve this problem, bone morphogenetic protein 4(BMP4) gene-enhanced adipose-derived stem cells(ADSCs) and CaAlg hydrogel scaffold was injected to the gap mosaicplasty left to promote the tissue integration and reduce the occurrence of "dead zone".

[Methods]

Porcine ADSCs were Isolated, cultured, and identified with multi-differentiation induce and specific stem cell markers. After transfection of BMP4 48 hours in vitro, the transfection efficiency was detected. The expression of BMP4 was observed by immunofluorescence staining. Expression and secretion of BMP4, collagen I and II, Sox9, ALP, Runx2 were detected by ELISA.

A miniature swine model of a huge 8.5mm-diameter 5mm-deep articular cartilage defect of the knee located at the femoral condyle was constructed. Three 3.5mm-diameter 5mm-deep osteochondral cylinders were removed from the femoral trochlea and transplanted into the osteochondal defect. CaAlg hydrogel scaffold combined with BMP4-ADSCs was injected to fill the "dead zone". 24 pigs were divided into three groups randomly. Group I received mosaicplasty only, Group II received mosaicplasty with CaAlg hydrogel injected into the „dead spaces‟ between the host osteochondral and osteochondral cylinder donor sites, and Group III received CaAlg hydrogel containing BMP4-ADSCs. Outcomes were evaluated 12 and 24

weeks post-operation. MRI, gross observation, histological staining (HE staining, toluidine blue staining), immunohistochemical staining (collagen type II), histological score and scanning electron microscope observation and biomechanical testing were evaluated. Data were expressed by mean ± standard deviation. The results were statistically analyzed by Mann-Whitney test with SPSS software. P<0.05 was statistically significant.

[Results]

In vitro experiment: P3 ADSCs were successfully isolated, cultured, and used for the following experiments. Under certain inducing conditions, ADSCs can differentiate into adipocytes, chondroblasts, and osteoblasts. The positive rates of CD90 and CD44 were 99.85% and 99.22% respectively. And the positive rate of CD34 representing blood derived surface marker molecules was 11.17%. The transfection efficiency of recombinant adenovirus vector with BMP4 gene was 98.86±0.74%. The expression of Bmp4 protein in ADSCs was detected by immunofluorescence after transfection 48h. BMP4 protein and collagen type I, type II, Sox9, ALP, and Runx2 were highly expressed in ADSCs transfected with BMP4 than in ADSCs by ELISA detection.

Group I: a clear boundary remained between transplanted osteochondral cylinders and surrounding tissues, the gap was filled with little fibrous scar tissue. Group II: osteochondral grafts were somewhat deformed and though neocartilage tissue was regenerated at the hydrogel injection site, cracks remained between osteochondral grafts and surrounding tissue. Group III: osteochondral tissue was completely integrated with a continuous intact surface. The neocartilage tissue exhibited toluidine blue staining, Masson staining, collagen type II immunohistochemical staining positive. The histological scores of the three groups were5.21±1.60,8.83±3.12,16.20±2.63 at 12 weeks; 7.24±1.32, 12.15±4.56, 21.42±1.35 at 24 weeks. Score of group III was significantly higher than the other two groups, which was close to normal cartilage. For the Group III, the contact stiffness and elastic modulus were close to the normal cartilage. The hardness was worse than that of the normal cartilage. But three parameters were significantly better than those of group I and group II.

[Conclusions]

Mosaicplasty and hydrogel containing BMP4-ADSCs can rapidly repair largecartilage defects with regeneration of native hyaline articular cartilage, repairing dead spaces between osteochondral grafts and donor-site defects.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124692
Appears in Collections:北京大学第三临床医学院_学位论文

Files in This Item:

There are no files associated with this item.


作者单位: 北京大学第三临床医学院

Recommended Citation:
陈临新. 骨形态发生蛋白4基因修饰的脂肪干细胞复合藻酸钙凝胶支架对骨软骨镶嵌成形术修复巨大软骨损伤的作用研究[D]. 北京大学第三临床医学院. 北京大学. 2015.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[陈临新]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[陈临新]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2017  北京大学医学部 - Feedback
Powered by CSpace