【作者】 齐义营；

【导师】 欧阳宏伟；

【作者基本信息】 浙江大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 背景:关节软骨损伤是骨科临床的常见疾病,而成熟关节软骨的自身修复能力较差,如果不给予治疗,持续发展会导致骨关节炎,给患者带来很大痛苦。传统治疗方法包括清创术、微骨折术、骨软骨移植等,能获得一定疗效但不能维持长期效果和治疗较大缺损。新兴的组织工程方法有可能治疗大面积的软骨缺损。支架和种子细胞是组织工程的两个重要因素,针对当今支架和种子细胞存在的一些局限,本研究设计了新型的具有疏松层和致密层的双层胶原支架并探求利用自体生长因子或胚胎干细胞来源的间充质干细胞修复软骨缺损。本研究分三个阶段:一、支架的研究:探求双层支架如何放置更利于修复软骨;二、支架复合生长因子的研究:探求双层胶原支架复合自体来源的富含血小板血浆是否能协同作用促进软骨的修复;三、支架复合种子细胞的研究:探求双层胶原支架复合胚胎干细胞来源的间充质干细胞修复软骨缺损的效率。第一部分双层胶原支架修复关节软骨缺损的研究目的:本研究制备具有疏松层和致密层的双层胶原支架修复软骨缺损,并比较支架如何放置更适合软骨的修复。方法:11只新西兰大白兔分为两组,A组:致密层向上组,B组:致密层向下组。在双膝髌股关节股骨髁部位做全层软骨缺损(厚度3mm,直径4mm),将支架分别按致密层向上、向下植入缺损处。分别于术后6周和12周处死动物,进行大体观察,组织学观察和力学测试。结果:术后6周和12周,与A组比较,B组在软骨缺损修复处有含量较多的透明软骨,有较高的GAG含量,6周后A、B组的组织学评分分别为5.7+/-2.1、4.3+/-1.8,12周后A、B组修复组织的组织学评分分别为10+/-1.4、8.7+/-2.1。12周的力学测试,A组弹性模量值0.169+/-0.057MPa,B组弹性模量值为0.133+/-0.023MPa,B组修复组织的弹性模量明显高于A组(P＜0.05)。结论:双层胶原支架能修复软骨缺损,其放置方式不同对修复结果产生影响,致密层向下放置的修复效果优于致密层向上的双层胶原支架。第二部分双层胶原支架复合自体来源的富含血小板的血浆修复软骨缺损的研究目的:微骨折术是一种利用释放骨髓来源的间充质干细胞来修复软骨损伤的一种常用的治疗方法,但是这种方法只能治疗较小的软骨缺损(＜2 cm~2)。本研究目的是研究自体来源的富含血小板的血浆(PRP)复合胶原支架是否能协同作用修复较大的软骨缺损。方法:38只新西兰大白兔膝关节随机分为3组,A组:对照组,B组:胶原支架组,C组:PRP复合胶原支架组。在双膝髌股关节股骨髁部位做全层软骨缺损(厚度3mm,直径4mm),分别给予未处理、植入胶原支架和植入胶原支架复合PRP。分别于术后6周和12周处死动物,进行大体观察,组织学观察和评分,以及力学测试。结果:在术后6周和12周,在这三组之中,C组软骨缺损处有含量最多的透明软骨,C组的GAG含量和组织学评分都高于其它两组,6周时A、B、C组的组织学评分分别为3.0+/-1.41、5.3+/-2.12、8+/-2.12,12周时分别为5.0+/-1.41、10.3+/-2.12、12.7+/-3.51。12周的力学测试表明C组和B组修复组织的弹性模量明显高于A组,B组为0.124+/-0.024MPa,C组为0.140+/-0.037MPa,A组为0.087+/-0.012 MPa(P＜0.05)。结论:自体来源的PRP复合胶原支架能协同作用,促进透明软骨的生成,促进软骨缺损的修复。本研究结果提示PRP复合胶原支架能修复较大面积的软骨缺损,从而有可能取代部分临床上治疗软骨缺损的自体软骨移植或骨软骨移植。第三部分双层胶原支架复合胚胎干细胞来源的间充质干细胞修复软骨缺损的研究目的:本研究利用新的种子细胞—胚胎干细胞来源的间充质干细胞(hESC-MSCs)复合双层胶原支架修复软骨缺损的效率。方法:16只SD大鼠分为两组,A:胶原支架组;B:胶原支架复合hESC-MSCs组。在双膝髌股关节股骨髁部位做全层软骨缺损(厚度2mm,直径2mm),分别给予植入胶原支架和植入胶原支架复合hESC-MSCs处理。于术后4周和8周处死动物,进行大体观察,组织学观察及力学测试。结果:术后4周和8周,B组软骨缺损修复处有含量较多的透明软骨,GAG含量高于A组。4周时A组弹性模量是0.031+/-0.0082MPa,B组是0.0322+/-0.014MPa,8周时A组弹性模量是0.035+/-0.012 MPa,B组是0.040+/-0.014 MPa。结论:hESC-MSCs复合双层胶原支架能存活并促进软骨的修复,hESC-MSCs有可能会成为软骨组织工程中新的种子细胞。更多还原

【Abstract】 Background:Articular cartilage injuries are common clinical diseases in orthopedics,but articular cartilage has very limited self-repair capability.Once injuried,if unreated,osteoarthritis will occur in longer time,which causes great pain for patients.Conventional methods for cartilage repair include debridement,microfracture,osteochondral graft and so on.While these methods can not maintain long-term therapeutic effect and can not treat larger cartilage defects.Tissue engineering method can treat larger cartilage defects.But current scaffolds and cells of cartilage tissue engineering are accompanied with disadvantages.To overcome those disadvantages,this study designed a novel bilayer collagen scaffold and explored autologous PRP as well as human embryonic stem cell derived mesenchymal stem cells(hESC-MSCs)to improve cartilage repair.This study included three parts.PartⅠ:To design a bilayer collagen scaffold and investigate their efficiency on cartilage repair;PartⅡ:To determine whether the collagen matrix with autologous platelet rich plasma(PRP)have synergistic effect to repair cartilage defects;PartⅢ:To determine the efficiency of the hESC-MSCs with bilayer collagen scaffold on cartilage repair.PartⅠ:Use of bilayer collagen scaffold for articular cartilage defects repair in a rabbit modelAIM:Bilayer scaffolds have been widely used for researches on cartilage repair.When compared to other kinds of bilayer scaffolds,collagen scaffold will have much perspective of clinical application because of its good biocompatibility of collagen.In this study we investigated the efficiency of this bilayer collagen scaffold and determine which layer should be adjacent to the surface of the subehondral boneMETHODS:Full-thickness cartilage defects(diameter=4 mm,thickness=3 mm)were made in the patellar grooves of New Zealand White rabbits(n=11).Bilayer collagen scaffold was implanted into the defect.Group A:dense layer was adjacent to the surface of the subchondral bone(n=11);Group B:loose layer was adjacent to the surface of the subchondral bone(n=11). Rabbits were sacrificed at 6 and 12 weeks after operation.The repaired tissues were processed for histology(n=3)at each time point and for mechanical test(n=5)at 12 weeks.RESULTHS:The results showed that at both 6 and 12 weeks,group B had more amounts of hyaline cartilage and had more glycosaminoglycans(GAGs)content.At 6 weeks,the histological scores of group A and B were 5.7+/-2.1、4.3+/-1.8 respectively,at 12 weeks,the histological scores of group A and B reached 10+/-1.4、8.7+/-2.1.The modulus of the repaired tissue in group B(0.133+/-0.023MPa)was significantly higher than that in groupA(0.169+/-0.057MPa)at 12 weeks post-surgery(P＜0.05).CONCLUSIONS:Bilayer collagen scaffold can repair cartilage defects.The dense layer to the bone marrow cavity is superior on cartilage regeneration compared to that of the loose layer.PartⅡ:Autologous platelet rich plasma(PRP)with bilayer collagen scaffold repair cartilage defects in a rabbit modelAIM:Microfracture is a routine therapy to release bone marrow from subchondral bone for in-situ cartilage repair.But it can only treat the small size of cartilage defects(＜2 cm~2).The study aims to investigate whether autologous platelet rich plasma(PRP)transplantation in collagen matrix can synergically improve the in-situ bone marrow initiated cartilage repair.METHODS:Full-thickness cartilage defects(diameter=4 mm,thickness=3 mm)were made in the patellar grooves of New Zealand White rabbits and treated with bilayer collagen scaffold (group B)and PRP with bilayer collagen scaffold(group C)and untreated(group A), respectively(n=11).The rabbits were sacrificed at 6 and 12 weeks after operation.The repaired tissues were processed for histology(n=3)at each time point and for mechanical test(n=5)at 12 weeks.RESULTS:The results showed that at both 6 and 12 weeks,group C had the most amounts of hyaline cartilage,which recovered larger cartilage area of surface at the cartilage defects.Also group C had higher histological scores and more glycosaminoglycans(GAGs)content than those in other two groups(p＜0.05).At 6 weeks,the histological scores of group A、B and C were 3.0+/-1.41、5.3+/-2.12、8+/-2.12 respectively,at 12 weeks,the histological scores of group A、B and C reached 5.0+/-1.41、10.3+/-2.12、12.7+/-3.51.The modulus of the repaired tissue in group B(0.124+/-0.024MPa)and group C(0.140+/-0.037 MPa)were significantly higher than that in groupA(0.087+/-0.012 MPa)(p＜0.05).CONCLUSIONS:Autologous PRP and bilayer collagen matrix synergistically stimulated the formation of cartilage tissues.The findings implicated that the combination of PRP with collagen matrix may repair certain type of cartilage defects larger than 2 cm~2 which currently require the complex autologous chondrocyte implantation(ACI)or osteochondral grafting.PartⅢ:Human embryonic stem ceil derived mesenehymal stem ceils(hESC-MSCs)with bilayer collagen scaffold repair cartilage defects in a rat modelAim:Chondrocytes and MSCs are often used as seed cells for cartilage tissue engineering.But they are accompanied with some disadvantages such as limited donor sources and lifespan.This study explored the use of hESC-MSCs for cartilage repair.METHODS:Full-thickness cartilage defects(diameter=2 mm,thickness=2 mm)were made in the patellar grooves of SD rats and treated with bilayer collagen scaffold(group A)and bilayer collagen scaffold with hESC-MSCs(group B),respectively(n=16).The rats were injected with cyclophosphamide(150mg/kg,ip)for immunosuppression and sacrificed at 4 and 8 weeks after operation.The repaired tissues were processed for histology(n=3)and for mechanical test(n=5)at each time point.RESULTS:The results showed that at both 4 and 8 weeks,group B had more amounts of cartilage and more glycosaminoglycans(GAGs)content than group A.The modulus of the repaired tissue in group B was higher than that in group A at 4 weeks(group A,0.031+/-0.0082MPa;group B, 0.0322+/-0.014MPa)and 8 weeks(group A,0.035+/-0.012 MPa;group B,0.040+/-0.014 MPa). CONCLUSIONS:hESC-MSCs with bilayer collagen scaffold can improve the cartilage formation of the defects and the hESC-MSCs can be alive,hESC-MSCs can be considered as candidate seed cells for cartilage tissue engineering.更多还原