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学科主题: 运动医学
题名:
异种异体脱矿皮质骨结合兔自体骨膜修复半月板缺失的实验研究
作者: 杨渝平
答辩日期: 2016-11-25
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: 半月板 ; 脱矿皮质骨 ; 骨膜 ; 异种异体 ; 同种异体 ; 移植
其他题名: An experimental study of the meniscus repair in rabbit with xenogenic demineralized cortical bone with lapine autologous periosteum
分类号: R684
摘要:

目的:探讨异种异体脱矿皮质骨(Demineralized Cortical Bone, DCB)结合兔自体骨膜修复半月板缺失的效果。

方法:36只新西兰白兔,随机分为3组,每组12只。全部采用手术制做内侧半月板缺失动物模型,并分别采用三种移植物进行修复:1、单纯采用牛DCB修复的作为对照组;2、新鲜同种异体半月板移植的作为同种异体组;3、牛DCB结合兔自体骨膜修复的作为实验组。分别在术后6周、12周和24周处死,观察三类不同移植物的大体和组织学转归以及对内侧间室软骨影响情况,并行生物力学检查。

结果:移植物大体观察:对照组移植物6周时全部从体部完全断裂,甚至脱落到关节腔,12周内被完全吸收;同种异体组6周时移植物完整,但12周和24周时,每组各有2例(50%)的半月板出现肉眼可见的明显体积缩小;实验组6周时仅有1例(25%)从体部断裂,其余愈合良好,12周和24周全部愈合良好;6周、12周和24周时,同种异体组和实验组移植物大体评分的差异无统计学意义(P>0.05)。移植物组织学HE染色观察:术后6周,对照组4例全部表现为无活细胞的骨基质,周围明显炎性细胞浸润,部分纤维化表现,12周内被完全吸收;同种异体组6周时移植物完整,但尖端变得圆钝,失去正常半月板的游离边缘形态,12周时半月板细胞中类似纤维母细胞的细胞数量明显增加,基质出现更加明显的松散,而且范围向游离缘侧扩展,24周时基质松散更加明显,游离缘仍然存在大量炎性细胞浸润;实验组6周时仅有1例(25%)从体部断裂,其余愈合良好,但牛DCB和自体骨膜间界限清晰,6-12周之间两者良好融合;除24周时同种异体组仍然有明显的异物反应和血管增生的表现,而实验组异物反应已经消失导致两组间评分差异有统计学意义以外(P<0.05),在其它时间两组间其它组织学评分差异均无统计学意义(P>0.05)。移植物天狼星红染色观察:同种异体组6周时中央区出现Ⅲ型胶原,12周和24周时,Ⅲ型胶原范围逐渐扩大甚至中央区出现胶原纤维松散断裂表现;实验组在12周和24周时,都以Ⅰ型胶原为主,可能有部分Ⅳ型胶原。移植物甲苯胺蓝染色观察:同种异体组12周和24周时都表现为明显的淡染;实验组12周和24周时表现为中心区骨化,周边可以见到大量的肥大软骨细胞。移植物生物力学检测:水合后牛DCB及同种异体组术后12周和24周时移植物的硬度和弹性模量都与正常兔半月板一致(P>0.05);实验组12周和24周的移植物硬度和弹性模量都要明显超过正常兔半月板,而且随着时间的延长,这种差异越来越明显(P<0.05)。软骨肉眼观察情况和组织学评分:同种异体组和实验组在术后6周、12周和24周时的评分都要优于对于对照组,但差异都没有统计学意义(P>0.05)。软骨甲苯胺蓝和Ⅰ型胶原染色观察:随着移植时间延长,三组都表现出甲苯胺蓝染色逐渐变浅、Ⅰ型胶原染色逐渐增多的趋势,而且都以对照组最为明显,同种异体组最不明显,实验组介于这两者之间。

结论:1、兔同种异体新鲜半月板移植后,可能因为免疫排斥,Ⅰ型胶原随着时间推移逐渐减少,基质逐渐松散,最终导致移植物失效。2、结合兔自体骨膜的牛DCB修复缺失半月板,移植后6-12周之间,会经过基质重塑,细胞长入等过程,最终与宿主之间有良好的组织相容性,形成半月板样结构,并对关节软骨具有一定的保护作用。3、结合兔自体骨膜的牛DCB与新鲜同种异体半月板移植修复缺失半月板对于关节软骨的保护作用相近。

英文摘要:

Objective

To investigate the result of meniscus repair in rabbit with xenogenic demineralized cortical bone (DCB) with lapine autologous periosteum.

Methods

36 adult New Zealand white rabbits that had undergone total medial meniscectomy were randomly divided into three groups: control group, allograft group and study group. We implanted three different types of grafts into their right knees: bovine DCB, fresh allograft meniscus and bovine DCB with lapine autologous periosteum, respectively. There were 12 rabbits in each group. After death, at 6 weeks, 12 weeks and 24 weeks, the grafts’ healing process were examined respectively with general observation and biomechanics and pathology examination. The cartilage of the medial compartment of each group was assessed by gross and semiquantitative histologic analysis as well.

Results

General observation of the grafts: which in control group were completely ruptured at 6 weeks, or even fall off into the joint, and were absorbed completely in 12 weeks. Which in allograft group were complete at 6 weeks. At 12 and 24 weeks, each group had 2 cases (50%) of the grafts appear obvious shrinkage to the naked eye. 3 cases (75%) healed well of study group at 6 weeks and all healed well at 12 and 24 weeks. At 6, 12 and 24 weeks, gross score differences of the allograft group and study group were no statistical significance (P > 0.05).

HE dyeing histology observation of grafts: All in control group were bone matrix without living cells and with obvious inflammatory cell infiltration at 6 weeks, and which were absorbed completely in 12 weeks. The grafts were complete in allograft group at 6 weeks but free edge became obtuse; at 12 weeks similar fibroblast cells of meniscus obviously increase the number of cells and matrix appeared more obvious loosely; matrix loose even more obvious at 24 weeks, and expanded to the free edge. Only 1 in study group was completely ruptured at 6 weeks, others healed well. But there were clear boundaries between bovine DCB and autogenous periosteum at 6 weeks, then good fusions came between 6 and 12 weeks.

Sirius red staining histology observation of grafts: Type Ⅲ collagen appeared in allograft group  at 6, 12 and 24 weeks, and the scope expanding gradually to the free edge and even appeared collagen fiber loose in central region.

Toluidine blue staining histology observation of grafts: Allograft group were acterized by obvious light dyeing at 12 and 24 weeks; study group at 12 and 24 weeks with central ossification, peripheral could see a lot of hypertrophic chondrocytes.

Biomechanical testing of grafts: Hardness and elastic modulus of the hydrated bovine DCB and the allograft were both consistent with that of normal lapine meniscus (p > 0.05). Which of study group at 12 and 24 weeks were significantly higher than that of the normal lapine meniscus, and over time, the differences were more and more obvious (P < 0.05).

Naked eye and histological grading of cartilage:At 6, 12 and 24 weeks, the gross scores of the allograft group and the study group were better than in the control group, but the difference had no statistical significance (p > 0.05).

Toluidine blue and typeⅠcollagen staining observation of cartilage: As the transplant time extended, all three groups showed the trend of increase of the toluidine blue staining became shallow gradually dyed, gradually, and with the control group was most remarkble, allograft group was the least significant, the study group was between the two.

 

Conclusions

1. Fresh lapine allograft meniscus transplantation, which may be due to immunological rejection, collagen type gradually reduce with time, matrix loose gradually, eventually lead to graft failure.

2. Between 6 and 12 weeks after transplantation, repairing a missing meniscus combined with lapine autologous periosteum, bovine DCB passed through matrix remodeling, cell ingrowth process, forming the meniscus-like tissue, finally had good histocompatibility with the host. And had certain protective effect for the articular cartilage.

3. Combined with lapine autologous periosteum, bovine DCB had similar effect for protection of articular cartilage with the fresh allograft meniscus transplantation to repair missing meniscus.

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/400002259/124739
Appears in Collections:北京大学第三临床医学院_学位论文

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作者单位: 北京大学第三临床医学院

Recommended Citation:
杨渝平. 异种异体脱矿皮质骨结合兔自体骨膜修复半月板缺失的实验研究[D]. 北京大学第三临床医学院. 北京大学. 2016.
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