异种脐血干细胞移植修复兔全层关节软骨缺损的初步研究

目的 :探讨人脐血干细胞对兔全层关节软骨缺损的修复作用及免疫反应。材料和方法 :取人脐带血中脐血干细胞及幼兔的骨髓基质细胞 ,体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 0只成年新西兰大白兔的股骨滑车关节面上造成直径 4.5mm深 3 .0mm的全层关节软骨缺损 ,将两种细胞 -PLA复合物分别植入关节软骨缺损处。植入异种脐血干细胞 -PLA复合物为实验组 ,植入同种异体骨髓基质细胞 -PLA复合物为阳性对照组 ,缺损不处理为阴性对照组。术后 6周、1 2周观察缺损修复情况、新生组织类型及有无免疫反应。结果 :脐血干细胞组 6周时标本为纤维组织修复 ,内有少量软骨细胞 ;1 2周时 40 %标本为软骨样组织修复 ,较薄 ;60 %标本为纤维组织修复。移植物周围无明显淋巴细胞聚集 ,部分滑膜有炎症反应。骨髓基质细胞组(阳性对照)为软骨样组织修复 ;滑膜无明显炎症反应。阴性对照组为纤维组织修复 ,无软骨形成。结论 :异种脐血干细胞移植修复软骨缺损优于缺损不处理组 (阴性对照) ,但明显差于同种骨髓基质细胞组 (阳性对照 )。脐血干细胞有可能成为软骨修复的新的种子细胞。由于种属差异的影响 ,脐血干细胞组可能存在免疫反应 ,结果需进一步研究     

组织工程重建兔颞下颌关节盘软骨

目的 应用组织工程学方法重建颞下颌关节盘软骨。方法 分离6只日本大耳白兔髁状突软骨细胞。进行细胞的微载体大规模扩增,将扩增后的软骨细胞接种于组织引导再生胶原膜,体外适当培养后植入4只同种成年兔皮下,植入后12周,对所获组织进行组织形态学观察。结果 髁状[突软骨细胞在胶原膜内生长良好,植入动物体内12周后可形成乳白色类软骨样组织,其表面光滑,有弹性。甲苯胺蓝染色,细胞周围基质呈异染性。结论 应用胶原膜结合软骨细胞共同培养,可形成软骨样组织,该方法将有可能成为软骨缺损及关节盘破损修复的有途径。     

骨髓间充质干细胞复合Ⅱ型胶原修复关节软骨缺损的组织学观察

目的 观察向软骨方向诱导分化的自体骨髓间充质千细胞(BMSCs)复合Ⅱ型胶原修复兔关节软骨缺损的质量.方法 扫描电镜观察Ⅱ型胶原结构.24只新西兰大白兔体外分离BMSCs,进行原代、传代培养,倒置显微镜观察.取第3代BMSCs,软骨诱导培养液培养14 d,调整细胞浓度为5×106/ml,与Ⅱ型胶原复合培养1周备用,并观察细胞在支架材料中的生长情况.于膝关节股骨滑车处制作一个直径为4 mm的圆柱形软骨缺损区,右侧植入复合培养1周的自体BMSCs和Ⅱ型胶原组织(实验组);左侧单纯植入Ⅱ型胶原(胶原对照组);另取12只,左右侧均不植入任何材料(空白对照组).术后2,4,8,12周取材,大体和组织学观察检测修复软骨的质量. 结果 Ⅱ型胶原呈网架状结构,孔隙大小在40～300μm之间,BMSCs在其中生长良好,软骨基质大量形成.实验组修复的关节面光亮、平整,镜下表现为透明样软骨,软骨细胞排列规律,与宿主软骨整合好;胶原对照组以纤维组织修复为主,与宿主软骨整合差;空白对照组未能修复软骨缺损.组织学评分实验组明显优于两对照组(P<0.05). 结论 自体BMSCs复合Ⅱ型胶原修复软骨缺损质量好,长期疗效需要进一步的观察.     

同种异体软骨细胞/聚乳酸复合物即时修复兔耳廓软骨缺损

 目的观察用同种异体软骨细胞/聚乳酸(Poly-DL-lactide,PDLLA)复合物在体内即时修复软骨缺损的能力.方法将兔耳廓软骨细胞体外分离消化,以PDLLA为支架,用软骨细胞/PDLLA复合物即时移植修复兔耳廓软骨缺损,对照组采用PDLLA,6、12、18周后观察软骨缺损修复情况.结果实验组移植后18周,软骨缺损愈合,修复软骨厚度均匀.对照组缺损区为条索状纤维组织修复,中央部凹陷.结论同种异体软骨细胞/PDLLA复合物即时修复软骨缺损是一种非常有前途的且适合临床应用的组织工程学方法.     

多聚乙醇酸-羟基磷灰石复合体为支架的骨髓基质细胞修复兔关节软骨缺损

目的 研究以多聚乙醇酸（polyglycolic acid，PGA）、羟基磷灰石（hydroxyapatites，HA）复合体为支架的骨髓基质细胞（bone marrow derived mesenchymal cells，BMSCs）修复兔膝关节软骨缺损修复效果。方法 自体BMSCs分别种植于PGA和HA支架后共同培养，生物胶粘连两种支架-细胞复合物形成BMSCs-PGA-HA复合体，植入兔膝关节软骨缺损模型。术后20，32周处死动物，标本行组织学检查及Ⅱ型胶原免疫组化染色。结果 BMSCs-PGA-HA复合体植入后形成稳定的透明软骨样修复组织及软骨下骨，该组织与周围正常软骨及软骨下骨融合良好，32周后软骨标本无明显退变。结论 以BMSCs为种子细胞、更符合缺损处生理结构的PGA-HA为支架的复合体，可修复关节软骨缺损，中长期效果值得肯定。     

骨髓基质细胞移植治疗骨缺损的实验研究

目的 探讨以Ⅰ型胶原海绵为载体的自体骨髓基质细胞移植治疗骨缺损的效果。方法 32只成年新西兰白兔随机分成Ⅰ、Ⅱ两组，分别自股骨大转子及胫骨结节抽取骨髓基质细胞进行培养，扩增后种植于Ⅰ型胶原海绵上，继续培养2周后植入1．5cm长桡骨缺损。Ⅰ组一侧桡骨缺损植入Ⅰ型胶原 自体骨髓基质细胞复合物(A处理)，对侧桡骨不做任何植入(B处理)。Ⅱ组一侧桡骨缺损做A处理，对侧桡骨缺损植入Ⅰ型胶原海绵(C处理)，分别于术后8周和12周取材，观察骨缺损的修复情况。结果 术后12周，A处理组全部骨性愈合，B处理组由纤维组织填充，C处理组有少量骨痂形成。结论 以Ⅰ型胶原海绵为载体的自体骨髓基质细胞移植能有效修复骨缺损。     

培养自体软骨细胞移植修复软骨缺损的分子生物学基础

创伤等原因所致的关节软骨缺损可引起患者疼痛,并导致滑膜炎和关节变性等,而软骨组织的自身修复能力又很差,缺损往往不能自行修复。目前临床上修复关节软骨缺损主要是自体软骨植入及软骨代用品植入,效果均不理想。近期研究表明,培养自体软骨细胞移植治疗软骨缺损取得了满意的疗效,这种方法还可在培养软骨细胞增殖和再分化的过程中调节基因表达[1]。笔者就自体软骨细胞移植治疗软骨缺损的分子生物学基础进行综述。一、关节软骨的分子生物学组成关节软骨(也称透明软骨)是覆盖在运动关节骨末端的一层质地坚韧的半透明组织,在膝关节厚度为1～5ｍ…     

聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损

目的：应用聚乙醇酸（ＰＧＡ）负载的兔软骨细胞培养移植修复同种异体关节软骨缺损．方法：应用在生物体内可降解吸收、纤维状多孔态的ＰＧＡ作为支架行兔软骨细胞培养．培养１４天后，软骨细胞在ＰＧＡ提供的三维空间中大量分裂、增殖并合成大量软骨基质，形成ＰＧＡ－软骨细胞复合体，然后利用该复合体移植修复同种异体兔膝关节全层软骨缺损，对侧膝关节作对照．术后行大体、组织学、电镜动态观察及修复组织厚度测定．结果：ＰＧＡ在术后８周完全降解吸收，实验侧与对照侧修复组织的厚度有显著性差异（Ｐ＜０．０１）；术后１６周在实验侧可见典型的软骨组织，电镜下为成熟的软骨细胞，而对照侧为纤维组织修复．结论：应用ＰＧＡ－软骨细胞复合体移植，可修复同种异体的兔关节软骨缺损，为临床治疗关节软骨缺损奠定了基础．     

猪腹膜脱细胞基质联合微骨折技术修复兔膝关节软骨缺损

目的:检测猪腹膜脱细胞基质(pig peritoneum-derived acellular matrix,PPAM)支架材料联合微骨折修复兔膝关节软骨缺损的效果。方法:采用CCK-8 Kit测定SD大鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMMSCs)在PPAM支架上的生长状态,Live/Dead染色观察细胞在PPAM支架上的活性。用直径4mm环钻造成深度约1.5 mm的新西兰大白兔膝关节软骨缺损,微骨折术后将PPAM支架植入软骨缺损处,单纯微骨折技术(Microfracture,MF)作为对照组,分别在第6周、12周和24周取材。大体观察、HE染色、甲苯胺蓝染色和II型胶原(COL II)免疫组化染色检测软骨缺损修复的效果。结果:CCK-8 Kit和Live/Dead染色结果显示PPAM支架支持细胞生长和增殖,无细胞毒性。大体观察和组织学检测显示PPAM联合MF修复软骨缺损能促进软骨缺损填充组织的质量,组织学评分优于单纯MF组。结论:PPAM支架是一种有良好生物相容性的天然材料来源组织工程材料,联合MF技术能促进关节软骨缺损的修复。     

应用组织工程化人工软骨修复羊关节软骨缺损的实验研究

目的 :探索以多孔磷酸三钙生物陶瓷材料为支架构建组织工程化软骨修复羊关节软骨缺损的可行性。方法 :实验分 3组。实验组 (n =12 ) :分离培养羊自体关节软骨细胞 ,采用微载体技术在旋转生物反应器内进行扩增 ,扩增后的软骨细胞接种到预制的 β_磷酸三钙 (β_TCP)多孔生物陶瓷材料上 ,细胞_材料复合体经体外孵育后 ,无菌条件下植入预制的羊前肢肱骨头关节面缺损处 ;单纯材料组 (n =12 ) :采用单纯 β_TCP材料修复羊关节软骨缺损 ;空白对照组 (n =4 ) :制备的羊关节软骨缺损区未做任何修复。术后 3和 6个月分别取材 ,进行缺损区组织学、组织化学和免疫组织化学分析。结果 :在实验组羊关节软骨缺损处表面肉眼可见透明软骨样组织形成。组织学检查发现 ,术后 3个月时材料降解明显 ,未降解吸收的材料孔洞内广泛分布着新生软骨组织 ,软骨细胞外基质丰富 ,Ⅱ型胶原染色阳性。至术后 6个月 ,支架材料几乎完全降解 ,缺损区被新生软骨组织所取代。在单纯材料组羊关节软骨缺损处术后 3个月时 ,可见从缺损区边缘有新生软骨组织向支架材料内长入 ,支架材料吸收明显。至术后 6个月 ,可见从缺损区边缘长入到支架材料内的新生软骨组织逐渐增多 ,但材料的中心部位未发现新生软骨形成。空白对照组羊关节软骨缺损区至术后 6     

组织工程软骨移植物修复兔关节软骨缺损

目的　观察组织工程软骨移植物修复兔关节软骨缺损的效果。　方法　经软骨起源诱导后的兔骨髓间质干细胞(mesenchymalstemcells,MSCs),与牛Ⅰ型胶原及人纤维蛋白相混合制成组织工程软骨移植物。60只5个月龄的日本大耳白兔均分为软骨移植物组、单纯载体对照组和空白对照组,观察各组修复兔股骨髁关节软骨全层缺损的效果。　结果　软骨移植物组12周时已形成正常厚度的软骨层及完整的软骨下骨板,O'drilscoll组织学评分18.22±2.45,Ⅱ型胶原含量97.9%,甲苯胺蓝变色反应表明其与周围正常软骨无明显区别,为透明软骨组织修复。而对照组12周时为纤维软骨修复,后期为纤维组织和板层骨修复。　结论　该组织工程软骨移植物作为软骨移植的替代物是可行的。     

兔外周血MSC复合同种异体脱钙皮质骨修复软骨缺损的研究

目的:比较外周血(peripheral blood,PB)与骨髓(bone marrow,BM)来源MSC复合脱钙皮质骨基质(demineralized cortical bone matrix,DCBM)修复兔膝关节软骨缺损的效果,为其进一步的临床应用提供实验参考。方法:获取分离培养的第三代兔PB-MSC和BM-MSC,以2×107/ml的细胞密度接种于DCBM上。20只成年新西兰兔,构建股骨远端滑车关节面中心部大面积全层软骨缺损模型,随机分为4组:I组:PB-MSC/DCBM复合物;II组:BM-MSC/DCBM复合物;III组:单纯DCBM支架组;IV组:空白缺损组。分别于术后24周、48周取材,通过大体观察、组织学评分、组织化学和免疫组织化学染色评价其在体内修复软骨的效果。结果:移植后24周PB-MSC/DCBM组:修复区由半透明组织填充,局部有小的浅的凹陷,表面光整,稍低于周围正常软骨,与周围组织连接连续。BM-MSC/DCBM组的大体观察类似I组。单纯DCBM组:表面光整,与周围组织连续性不如I、II组。空白缺损组:仍有一大的缺损。48周PB-MSC/DCBM组和BM-MSC/DCBM组,缺损区基本不可见,充填好,表面光滑,基本和周围正常组织无界限,与周围软骨连续性好,软骨细胞外基质表达较多。组织学评分显示:在24周和48周两个时间点,PB-MSC/DCBM组和BM-MSC/DCBM组比较组间无差异,均显著高于单纯DCBM组和空白缺损组。结论:PB-MSC具备与BM-MSC相似的修复兔关节软骨缺损的能力。     

Fresh osteochondral allografting

The treatment of articular cartilage defects in the knee is a difficult challenge. Fresh, small-fragment osteochondralallografting is a technique involving the transplantation of articular (hyaline) cartilage into the defective joint surface. The graft, a composite of living cartilage and a thin layer of underlying subchondral bone, provides a mature matrix with viable chondrocytes along with an osseous component that provides a surface for fixation and integration with the host. Fresh allografting is particularly useful in larger lesions (greater than 2 cms) or when associated osseous defects are present. Clinical experience with fresh osteochondral allografts now extends over 2 decades. Up to 90% of individuals treated for femoral condyle lesions are improved. The allograft tissue appears well tolerated by the host, with documented long-termsurvival of chondrocytes and intact matrix. Successful clinical outcomes have established fresh osteochondrall allografting as an appropriate alternative in the treatment of chondral and osteochondral lesions of the knee.     

Natural history of bone bruises after acute knee injury: clinical outcome and histopathological findings

The purpose of this paper is to review the scientific literature on the natural history of bone bruises and the experimental studies regarding the histopathological effects of impaction load on articular cartilage and subchondral bone. Bone bruises with subchondral or osteochondral injuries, or geographic bone bruises seemed to be persistent for years after trauma on MRI. Biopsy samples of the articular cartilage overlying the bone bruise lesions showed degeneration or necrosis of chondrocytes and loss of proteoglycan. Experimental studies using a single impact load revealed chondrocytes death, alteration of the mechanical properties of cartilage explants and/or an increase in the thickness of subchondral bone. These data are indicative of a significant injury to normal articular cartilage homeostasis, and support the suggestion that severe bone bruise is a precursor of early degenerative changes. We recommend delaying return to full weightbearing status when a severe bone bruise is detected to prevent further collapse of subchondral bone and further aggravation of articular cartilage injury.     

Mesenchymal stem cell-based therapy for cartilage repair: a review

Articular cartilage injury remains one of the major concerns in orthopaedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques. The purpose of this paper is to review the literature on MSC-based cell therapy for articular cartilage repair to determine if it can be an alternative treatment for cartilage injury. MSCs retain both high proliferative potential and multipotentiality, including chondrogenic differentiation potential, and a number of successful results in transplantation of MSCs into cartilage defects have been reported in animal studies. However, the use of MSCs for cartilage repair is still at the stage of preclinical and phase I studies, and no comparative clinical studies have been reported. Therefore, it is difficult to make conclusions in human studies. This requires randomized clinical trials to evaluate the effectiveness of MSC-based cell therapy for cartilage repair.     

移植基质诱导的自体软骨细胞修复关节软骨缺损临床研究

 目的 探讨基质诱导的自体软骨细胞移植修复关节软骨缺损的方法与疗效.方法 2004年11月～2006年11月,对7例膝关节软骨炎患者行关节镜取软骨、基质诱导自体软骨细胞移植(Matrix-induced Autologous chondrocyte implantation,MACI)膜植入术.对患者行MRI检查确定损伤位置,并进行IKDC2000评分.术后按照特定的康复计划进行循序渐进的功能锻炼.结果 随访时间6个月到24个月.术后半年多数患者各项症状逐渐消失,IKDC2000评分大部分增高.复查MRI和关节镜,显示原来缺损的关节软骨已基本修复,并伴有软骨下骨的修复.结论 与传统自体软骨细胞移植(Autologous chondrocyte implantation,ACI)技术相比,利用MACI技术修复软骨缺损具有术后恢复时间短、操作简便、创伤小、生成更多透明软骨等优点,具有良好的应用前景.     

Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo

Purpose

The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested.

Methods

Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo.

Results

After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants.

Conclusion

The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.     

自体MSCs/Chondro-Gide工程化软骨再生修复技术的大动物实验

目的探索自体MSCs/Chondro-Gide工程化软骨修复技术用于软骨再生治疗的可行性。方法 12只山羊随机分为实验组、自体基质诱导软骨再生（autologous matrix-induced chondrogenesis,AMIC）治疗组及空白对照组,实验组采用自体MSCs/Chondro-Gide工程化软骨修复技术修复软骨缺损;AMIC治疗组采用AMIC技术,空白对照组不植入任何材料。术后8周行大体观察、关节镜检查、影像学检测、组织学检测、改良Wakitani法评分,评价其对关节软骨缺损的再生修复效果。结果 8周大体观察实验组软骨缺损的修复及整合良好;MRI及关节镜提示软骨缺损修复表面光滑,弹性可;组织学检测修复区域有较多幼稚软骨细胞;AMIC组可见修复区为纤维组织修复;空白组为少量纤维组织覆盖缺损底面。8周及16周改良Wakitani评分示：实验组修复效果优于AMIC组及空白对照组。结论自体MSCs/Chondro-Gide工程化软骨修复技术创伤小,操作简便,能显著促进关节软骨缺损的再生修复,具有较高的科学价值及临床应用前景。     

Biochemical and biomechanical properties of lesion and adjacent articular cartilage after chondral defect repair in an equine model

BACKGROUND: Chondral defects may lead to degradative changes in the surrounding cartilage, predisposing patients to developing osteoarthritis. PURPOSE: To quantify changes in the biomechanical and biochemical properties of the articular cartilage adjacent to chondral defects after experimental defect repair. STUDY DESIGN: Controlled laboratory study. METHODS: Specimens were harvested from tissue within (lesion), immediately adjacent to, and at a distance from (remote area) a full-thickness cartilage defect 8 months after cartilage repair with genetically modified chondrocytes expressing insulin-like growth factor-I or unmodified, control chondrocytes. Biomechanical properties, including instantaneous Young's and equilibrium aggregate moduli, were determined by confined compression testing. Biochemical properties, such as water and proteoglycan content, were also measured. RESULTS: The instantaneous Young's modulus, equilibrium modulus, and proteoglycan content increased, whereas water content decreased with increasing distance from the repaired lesion. The instantaneous Young's and equilibrium moduli of the adjacent articular cartilage were 80% and 50% that of remote area samples, respectively, whereas water content increased 0.9% and proteoglycan content was decreased by 35%. No significant changes in biomechanical and biochemical properties were found either in the lesion tissue or in adjacent cartilage with genetic modification of the chondrocytes. CONCLUSION: Articular cartilage adjacent to repaired chondral defects showed significant remodeling 8 months after chondral defect repair, regardless of whether genetically modified or unmodified cells were implanted. CLINICAL RELEVANCE: Changes in the biochemical and biomechanical properties of articular cartilage adjacent to repaired chondral defects may represent remodeling as part of an adaptive process or degeneration secondary to an altered distribution of joint forces. Quantification of these changes could provide important parameters for assessing progress after operative chondral defect repair.