Preclinical and clinical data for the use of mesenchymal stem cells in articular cartilage tissue engineering

Introduction: With an ageing population, the prevalence of osteoarthritis (OA) has increased. Mesenchymal Stem Cells (MSCs) have been proposed to be an attractive alternative candidate in the tissue engineering of articular cartilage primarily due to its abundant source, reduced cartilage donor site morbidity, and strong capacity for proliferation and potential to differentiate toward a chondrogenic phenotype. Areas covered: A current overview of human, in vivo, and in vitro evidence on the use of MSCs in cartilage tissue engineering. Expert opinion: We demonstrate robust evidence that MSCs have the potential to regenerate articular cartilage. We also identify the complexity of designing a suitable preclinical model and the challenges in considering its clinical application such as type of MSC, scaffold, culture construct and the method by which growth factors are delivered. Of great interest is further characterization of the factors that may prevent MSC-derived chondrocytes to undergo premature hypertrophy and to understand what enables the terminal developmental pathway for permanent hyaline cartilage regeneration. Despite this, there is an abundance of evidence suggesting that MSCs are a desirable cell source and will have significant impact in tissue engineering of cartilage in the future.     

Preclinical and clinical data for the use of mesenchymal stem cells in articular cartilage tissue engineering

Introduction: Hemophilia B, the deficiency or complete absence of coagulation factor IX (FIX), affects an estimated 80,000 people throughout the world. Some of these individuals are managed with prophylaxis, which involves the intravenous infusion of FIX concentrate two to three times weekly to prevent bleeding. Because FIX prophylaxis remains underutilized, patients with hemophilia B are at risk for bleeding that may be severe and potentially life- or limb-threatening, and they may experience arthropathy resulting from recurrent hemarthroses.

Areas covered: This review focuses on recent advances in therapeutic protein fusion technology as they apply to FIX deficiency. The National Library of Medicine Medline database was searched for articles containing the term ‘Fc fusion proteins’.

Expert opinion: Genetically engineered recombinant FIX fused to the Fc portion of immunoglobulin significantly extends FIX half-life, thereby decreasing the frequency of prophylactic infusions. This in turn may increase the adoption of, and adherence to, prophylaxis, leading to better outcomes for hemophilia B patients.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。方法：由第一作者检索1990至2011年PubMed数据库（http：／／www．ncbi．nlm．nih.gov／PubMed）有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,genetherapy,mesenchymalstemcells,tissueengineering,bioactivefactor,vector”。共纳入15篇文献归纳总结。结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

Current concepts in stem cell therapy for articular cartilage repair

Introduction: Hyaline articular cartilage is the connective tissue responsible for frictionless joint movement. Its degeneration ultimately results in complete loss of joint function in the late stages of osteoarthritis. Intrinsic repair is compromised, and cartilage tissue regeneration is difficult. However, new options are available to repair cartilage tissue by applying ESCs, MSCs and CPCs.

Areas covered: In this review, the authors shed light on the different concepts currently under investigation for cartilage repair.

Expert opinion: So far, there is no way to derive a chondrogenic lineage from stem cells that forms functional hyaline cartilage tissue in vivo. One alternative might be to enhance the chondrogenic potential of repair cells, which are already present in diseased cartilage tissue. CPCs found in diseased cartilage tissue in situ are biologically driven toward the osteochondrogenic lineage and can be directed toward chondrogenesis at least in vitro.     

关节软骨重建与间充质干细胞

目的:间充质干细胞是软骨组织工程中理想的种子细胞,但干细胞向软骨细胞分化的机制尚不十分清楚,真正的临床应用仍面临许多问题。就干细胞的来源,定向诱导分化,以及体内外构建组织工程化软骨等方面的研究进行综述,为以间充质干细胞为基础的关节软骨重建提供参考。资料来源:应用计算机检索1991-01/2004-06Ovid全文数据库和Medline数据库相关文章,限定文章语言种类为English。检索词“tissueengineering,stemcells,cartilage,articular”,以及维普系列数据库及http://www.wanfangdata.com.cn,并限定文章语言种类为中文,检索词“组织工程,关节软骨,间充质干细胞”。资料选择:纳入条件:①所选文献主要是间充质干细胞为种子细胞的软骨组织工程方面的实验性研究及临床研究。②遵循随机对照的原则。③对研究中是否采用盲法无严格的限制。排除条件:①对单纯论述间充质干细胞或组织工程化软骨方面的文献以及个案报道文献。②综述文献。资料提炼:共收集到30篇相关文献,17篇符合上述标准。资料综合:在体内外,不同来源的间充质干细胞可以经诱导,分化成软骨组织。诱导物为转化生长因子超家族成员,在三维立体条件下,高细胞密度(1×109L-1)以及适宜的培养时间,机械刺激有利于软骨的形成,并且在临床应用上取得满意的结果,修复组织的组织学及关节镜评分均优于无细胞移植的对照组。结论:间充质干细胞在体内外可以诱导形成关节软骨,有望应用于临床上关节软骨损伤的重建。     

骨髓间充质干细胞定向诱导移植修复关节软骨

背景:骨髓间充质干细胞在不同诱导条件下具有向中胚层组织细胞如成骨细胞、成软骨细胞、肌细胞、脂肪细胞等分化的能力.目的:验证用组织工程方法诱导分化骨髓间充质干细胞修复兔关节软骨损伤的效果.设计、时间及地点:随机对照动物实验,于2005-05-03/2007-12-30在沈阳医学院临床中心实验室完成.材料:20只两三月龄的健康新西兰白兔,雌雄不限.方法:①诱导分化体外培养的兔骨髓间充质干细胞.实验组加入地塞米松、碱性成纤维细胞生长因子、维生素C培养1周,再将转化生长因子β替换碱性成纤维细胞生长因子培养3周;以不加诱导剂细胞做对照.②取20只兔.建立膝关节软骨缺损模型,随机分为3组.实验组10只膝关节内植入经诱导的骨髓间充质干细胞;对照组植入未经诱导的骨髓间充质干细胞:空白对照组植入生理盐水.分别于术后2,4,6,8周时处死实验组处死2只,对照组和空白对照组各处死1只进行各项指标检测.主要观察指标:①细胞形态学.②碱性磷酸酶活性的测定.③大体标本观察.④X射线观察.⑤组织切片观察.结果:①经诱导的骨髓间充质干细胞,细胞形态发生明显变化,逐渐由长梭形变为多角形,类似于软骨细胞样形态.②骨髓间充质干细胞经诱导4周后,其碱性磷酸酶活性明显增强(P＜0.05).③术后8周,实验组标本修复组织表面光滑,与周围软骨间界限模糊不清:X射线表现为关节间隙变宽,软骨下骨质囊变得到改善:组织切片观察显示与正常软骨细胞基本上一致.结论:自体间充质干细胞移植可修复关节软骨损伤.     

组织工程化软骨细胞和骨髓间充质干细胞用于修复同种异体关节软骨缺损

背景:关节软骨几乎没有自身修复的能力,目前临床大多采用自体或异体软骨移植修复、软骨膜或骨膜移植修复、软骨细胞移植修复。由于自体软骨来源有限,异体软骨又存在慢性免疫排斥反应,最终可能导致预后不佳;软骨膜或骨膜移植修复的软骨易于退化,导致修复效果不佳。目的:总结组织工程化软骨细胞、骨髓间充质干细胞及两者共培养对同种异体软骨缺损修复作用的研究现状。方法:应用计算机检索PubMed数据库及中国期刊网全文数据库1994-01/2012-01有关组织工程化软骨细胞和骨髓间充质干细胞用于修复同种异体关节软骨缺损方面的文章,英文检索词为"cartilage defect,allograft,chondrocyte,mesenchymal stem cells,bone marrow mesenchymal stem cells",中文检索词为"软骨缺损,同种异体移植,软骨细胞,骨髓间充质干细胞"。排除重复性及非中英文语种研究,共保留35篇文献进行综述。结果与结论:随着体外细胞培养方法的不断改进,现已能够把软骨细胞从坚韧的软骨中分离出来,并获得大量高纯度的软骨细胞并繁殖出新生软骨细胞。软骨细胞培养增殖能力低,传代培养容易引起老化和去分化;而成体骨髓中骨髓间充质干细胞含量少,随传代次数的增多成软骨潜能明显降低。骨髓间充质干细胞和软骨细胞共培养,两种细胞相互促进增殖和分化,作为种子细胞可减少软骨细胞增殖传代次数并节省软骨细胞数量,与组织工程支架材料复合能有效修复关节软骨缺损。     

间充质干细胞在软骨组织工程中的作用研究

间充质干细胞是一类存在于人体多种组织中的非造血多潜能细胞,可以自我更新或分化为骨、软骨或脂肪组织等多种细胞。近年来间充质干细胞在软骨组织工程中的研究取得了巨大进展,本文就间充质干细胞诱导成软骨细胞的因素及其在关节软骨损伤、免疫性疾病、组织修复和软骨组织工程中的应用研究等方面展开综述。     

间充质干细胞在关节软骨组织工程中的应用

背景：组织工程技术的发展为关节软骨缺损修复和功能重建提供了新的方法和思路。目的：探讨以间充质干细胞作为种子细胞在关节软骨组织工程中的应用和研究进展。 方法：由第一作者检索 PubMed 数据库中2000-01-01/2014-09-30有关间充质干细胞和关节软骨组织工程的文献,检索词为“articular cartilage defects, cartilage tissue engineering, mesenchymal stem cel s”。共检索到70篇相关文献,对其中49篇文献进行综述。 结果与结论：关节软骨缺损自身修复能力很有限,目前的临床治疗手段无法达到满意修复,而组织工程的发展为解决这个问题提供了新思路。在种子细胞选择方面,软骨细胞去分化能力有限,胚胎干细胞受到伦理、法律等方面的制约,而间充质干细胞因其自体来源、易扩增、具有软骨分化潜能而受到广泛重视。但目前应用组织工程方法修复关节软骨缺损的效果存在一定的争议,主要是远期功能距离临床应用存在一定差距,在修复组织结构和生物力学方面还需要进一步研究。     

Use of stem cells in the biological repair of articular cartilage

Importance of the field: Articular cartilage is avascular, aneural, and renowned for its poor capacity to repair after damage. For decades scientists and clinicians have deliberated over the potential to repair or regenerate articular cartilage and to date many techniques have been used in an attempt to create the best possible repair tissue.

Areas covered in this review: This review article summarises surgical interventions that have been developed since the late 1940's; covering conservative strategies, invasive techniques and touching upon latest advancements involving stem cells and tissue engineering.

What will the reader gain: The reader will gain a sound understanding into the history and background of strategies that have developed in attempts to reverse clinical symptoms of damaged or diseased articular cartilage. The article provides an insight into the plethora of potential repair mechanisms, and reviews future developments involving stem cells and biomaterials.

Take home message: Although work is still in its infancy, the use of stem cells in the biological repair of articular cartilage provides a promising outlook onto future developments; advancing from strategies and techniques that are already in use.     

骨髓间充质干细胞复合多肽凝胶及成软骨生成因子修复兔关节软骨缺损

背景：多肽水凝胶因为其具有良好的可塑型性,能够与损伤部位很好的无缝隙结合,所以采用该材料作为支架是骨、软骨组织工程中一种可行的探索。目的：骨髓间充质干细胞联合新型可注射多肽凝胶及成软骨生成因子修复兔关节软骨缺损,观察其修复效果。方法：首先分离培养兔骨髓间充质干细胞,兔左侧膝关节处制备直径5 mm,深3 mm的全层骨-软骨缺损模型;右侧造模后空置作为对照。实验分为3组,单纯自组装多肽凝胶移植组,自组装多肽凝胶+成软骨因子组和自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组。采用的成软骨因子包括转化生长因子β1,地塞米松和胰岛素样生长因子1,三者混合后加入到自组装多肽凝胶或骨髓间充质干细胞中。于处理后12周时处死动物行大体及组织学观察、X射线摄片、免疫组织化学法进行组织学评分评估修复情况。结果与结论：单纯自组装多肽凝胶移植在12周后显示出非常好的修复效果,可见番红O染色,Ⅱ型胶原蛋白免疫组织化学染色强度以及组织学评分明显高于其他组(P <0.05)。自组装多肽凝胶+成软骨因子组修复效果较好,与自组装多肽凝胶组相似,但其修复区域蛋白聚糖表达比对照组明显升高(P <0.01)。自组装多肽凝胶+成软骨因子+骨髓间充质干细胞组修复效果不佳,12周未能完全修复缺损区域,与单纯自组装多肽凝胶组比较骨赘的形成有所增加。结果表明,单纯自组装多肽凝胶能够在原位修复骨软骨缺损并促进软骨修复,提示以自组装多肽凝胶支架移植有望提高目前修复软骨缺损的效果。     

滑膜间充质干细胞修复软骨损伤：具有临床转化机制的话题

背景：软骨损伤目前仍然是临床上难以完全治愈的一大难题。近来,滑膜间充质干细胞的发现为该病变的治疗带来了新的希望。 目的：综合近几年文献探讨滑膜间充质干细胞的特性、培养条件、临床前及临床研究等关于软骨修复的研究进展。 方法：应用计算机检索1993年1月至2013年5月PubMed数据库和SpringerLink数据库中的相关文章,检索词为＂synovial mesenchymal stem cells, cartilage repair＂,并参阅其他相关的著作及高影响因子的相关文献. 结果与结论：最终纳入符合标准的文献37篇。滑膜间充质干细胞较之其他间充质干细胞具有更强的增殖、集落形成和软骨化能力。骨关节炎、类风湿关节炎等疾病可影响其细胞特性。已有大量文献集中于其体外细胞培养及动物体内细胞移植方面的研究。然而,该治疗方法的研究尚处于初步阶段。关于其细胞鉴别特性、理想培养条件及高质量临床研究方面的报道仍相当缺乏。总而言之,尽管研究有待深化,滑膜间充质干细胞仍不失为一种未来修复软骨损伤领域有前景的细胞资源。     

富血小板血浆及脐带间充质干细胞修复软骨损伤

背景：研究者直接将富血小板血浆作为支架材料与骨髓基质干细胞、软骨细胞等复合后体外培养发现软骨细胞在富血小板血浆三维支架呈现增殖生长,骨髓基质干细胞在增殖的同时有向软骨细胞分化的倾向。目的：观察富血小板血浆和人脐带间充质干细胞对受损软骨修复的影响。方法：正常健康新西兰大白兔40只,制备兔软骨损伤模型。2次离心法制备富血小板血浆,制备3代人脐带间充质干细胞。随机将动物分为4组,造模后生理盐水组关节腔一次性注入生理盐水0.5 mL;富血小板血浆组注入12.5%富血小板血浆0.5 mL;人脐带间充质干细胞组注入1×107人脐带间充质干细胞0.5 mL;富血小板血浆(12.5%)联合人脐带间充质干细胞(1×107)组注入两种物质0.5 mL。造模后第12周,大体观察软骨损伤修复情况;苏木精-伊红染色光镜下观察损伤部位细胞修复情况;根据O’Driscol组织学评分标准对造模后第12周切片进行组织学评分。结果与结论：软骨损伤后大体观察、苏木精-伊红染色组织学观察及组织学评分均显示富血小板血浆组、人脐带间充质干细胞组、富血小板血浆联合人脐带间充质干细胞组对软骨损伤的修复效果优于生理盐水组,差异有显著性意义(P<0.05),富血小板血浆组、富血小板血浆联合人脐带间充质干细胞组的修复效果优于人脐带间充质干细胞组,差异有显著性意义(P<0.05)。结果说明富血小板血浆、人脐带间充质干细胞均能促进软骨损伤的修复,而且富血小板血浆、富血小板血浆联合人脐带间充质干细胞较单独应用人脐带间充质干细胞效果好。     

微重力三维动态诱导骨髓间充质干细胞复合可注射型支架材料Pluronic F-127修复关节软骨缺损

目的:利用三维动态诱导的同种异体非软骨来源种植细胞,复合可注射型支架材料聚氧乙烯聚氧丙烯共聚物Pluronic F-127修复关节软骨缺损。方法:实验于2005-06/2006-03在哈尔滨医科大学附属第一医院动物实验中心完成。①选取2个月龄新西兰大白兔27只,3只用于骨髓间充质干细胞的分离,剩余24只随机数字表法分为诱导细胞复合支架组、单纯支架组、空白对照组,8只(16膝)/组。可注射型支架材料Pluronic F-127为白色粉沫状,无味,其水溶液在低温4℃呈液态,37℃形成固态凝胶(sigma公司,产品批号P2443)。②兔麻醉后穿刺抽取胫骨骨髓,分离培养骨髓间充质干细胞,取3代细胞进行实验。③收集细胞,离心成团,分4组不同诱导条件。三维动态培养组:10g/L藻酸钠溶液洗涤并重新悬浮细胞,细胞浓度为5×1010L-1,滴入200mmol/L氯化钙溶液,立即形成藻酸钙凝胶微球,细胞被悬浮固定于球内部,静止5min,取出凝胶微球,置于旋转式细胞培养系统,微重力条件下动态诱导。二维动态培养组:细胞直接接种于旋转式细胞培养系统,微重力条件下动态诱导。三维静态培养组:藻酸钙凝胶微球悬浮细胞接种于培养瓶静态培养。二维静态培养组:细胞直接接种于平面培养瓶静态培养。各组细胞诱导培养2周,制备切片,分别行甲苯胺蓝染色及Ⅰ、Ⅱ型胶原免疫组织化学染色。柠檬酸钠溶液溶解藻酸钙凝胶微球,测定各组胶原和蛋白多糖含量。④各组兔的双膝均制备膝关节全层软骨缺损模型。诱导细胞复合支架组选取诱导分化最佳的骨髓间充质干细胞与25%可注射型支架材料Pluronic F-127在低温下混匀,重悬,细胞终浓度为5×1010L-1,注入股骨内髁软骨缺损区。单纯支架组只将25%Pluronic F-127注入股骨内髁软骨缺损区。空白对照组关节软骨缺损区不作任何处理。各组分别于术后4,8,12,24周取材,大体及镜下观察关节软骨表面缺损修复情况,同时进行组织学评分。结果:24只兔全部进入结果分析。①骨髓间充质干细胞体外诱导培养及鉴定结果:藻酸钠接触钙离子迅速形成透明凝胶微球,有光泽,凝胶微球中的细胞呈球形,核仁清晰,与正常软骨细胞立体结构相似。培养过程中各组细胞甲苯胺兰染色均呈阳性,并表达Ⅱ型胶原,但无明显Ⅰ型胶原表达。②不同体外诱导分化条件下细胞生化指标的表达:三维、二维动态培养组的胶原和蛋白多糖含量均明显高于三维、二维静态培养组,且以三维动态培养组效果最佳[(0.078±0.002),(0.048±0.002),(0.035±0.001)A,P<0.05;(0.111±0.003),(0.069±0.003),(0.058±0.002)A,P<0.05]。③关节软骨缺损修复情况:诱导细胞复合支架组术后12周修复软骨表面光滑,质地坚硬,为透明软骨组织结构,与周围软骨结合紧密,至24周仍保持透明软骨形态;而单纯支架组、空白对照组均未被修复。术后4,8,12,24周,诱导细胞复合支架组关节软骨组织学评分均优于单纯支架组、空白对照组(P<0.05),且术后时间越长,修复效果越佳。结论:微重力条件下三维动态诱导骨髓间充质干细胞可提高分化质量,获得的种植细胞复合可注射型支架材料修复关节软骨缺损能够取得稳定的长期修复效果。     

骨髓间充质干细胞与关节软骨细胞Transwell共培养诱导形成软骨

背景:研究证实,关节软骨细胞具有分泌诱导因子促进骨髓间充质干细胞向关节软骨细胞分化的能力,但至今未见两种细胞运用Transwell共培养诱导骨髓间充质干细胞形成软骨的报道.目的:观察在Transwell共培养系统中共培养后,关节软骨细胞诱导骨髓间充质干细胞向关节软骨样细胞转化的能力.方法:骨髓间充质干细胞来源于4周龄SD大鼠的股骨及胫骨干骨髓,关节软骨细胞来源于4周龄SD大鼠的正常股骨头表面的关节软骨.分别吸取第3代骨髓间充质干细胞和关节软骨细胞,按1:1的细胞比例分别植于Transwell共培养系统中,下室植入骨髓间充质干细胞,上室植入关节软骨细胞.同时设置相同浓度单纯骨髓间充质干细胞对照组,分别在含胎牛血清的DMEM培养基中培养,相差显微镜下观察细胞的增殖和基质合成情况,并对各组细胞爬片进行Ⅱ型胶原免疫组化染色及氨基聚糖含量检测.结果与结论:共培养组骨髓间充质干细胞数量增多,细胞外基质合成丰富,其基质能被Ⅱ型胶原免疫组化染色,细胞染色呈现黄色.氨基聚糖含量随着诱导时间增加而增多.提示关节软骨细胞分泌物具有促进骨髓间充质干细胞向关节软骨样细胞转化的能力,与此同时,骨髓间充质干细胞还能分泌促进组织细胞修复的细胞因子,使共培养中的软骨细胞功能得以加强.由此可见,软骨细胞与骨髓间充质干细胞共培养诱导具有独特的优势.     

The importance of optimal drug sequencing in metastatic colorectal cancer: biological rationales for the observed survival benefit conferred by first-line treatment with EGFR inhibitors

Introduction: Use of both the vascular endothelial growth factor (VEGF) inhibitor bevacizumab and the epidermal growth factor receptor (EGFR) inhibitors cetuximab and panitumumab as potential first-line therapies for patients with RAS-wild-type metastatic colorectal cancer presents clinicians with an important decision. We review clinical data evaluating first-line treatment with EGFR inhibitors. Additionally, by undertaking an integrated ‘bench-to-bedside’ approach, we provide potential models, testable hypotheses and biological rationales that might account for these clinical observations.

Areas covered: A literature search encompassing PubMed and the ASCO/ESMO websites was undertaken in October 2014. Search terms included ‘colorectal cancer’, ‘cetuximab’, ‘panitumumab’ and ‘bevacizumab’.

Expert opinion: A number of clinical studies indicate a survival benefit for patients receiving EGFR inhibitors in combination with chemotherapy in the first-line setting, relative to both chemotherapy alone and VEGF inhibitors plus chemotherapy. Existing preclinical and clinical data suggest that a biological basis exists for providing RAS-wild-type patients with first-line EGFR inhibitors, followed by second-line VEGF inhibitors. More specifically, first-line treatment with EGFR inhibitors may elicit unique biological changes that sensitize tumors to subsequent lines of therapy; conversely, first-line treatment with VEGF inhibitors may elicit biological changes that desensitize tumors to subsequent lines of therapy.     

Gene-based approaches for the repair of articular cartilage

Gene transfer technology has opened novel treatment avenues toward the treatment of damaged musculoskeletal tissues, and may be particularly beneficial to articular cartilage. There is no natural repair mechanism to heal damaged or diseased cartilage. Existing pharmacologic, surgical and cell based treatments may offer temporary relief but are incapable of restoring damaged cartilage to its normal phenotype. Gene transfer provides the capability to achieve sustained, localized presentation of bioactive proteins or gene products to sites of tissue damage. A variety of cDNAs have been cloned which may be used to stimulate biological processes that could improve cartilage healing by (1) inducing mitosis and the synthesis and deposition of cartilage extracellular matrix components by chondrocytes, (2) induction of chondrogenesis by mesenchymal progenitor cells, or (3) inhibiting cellular responses to inflammatory stimuli. The challenge is to adapt this technology into a useful clinical treatment modality. Using different marker genes, the principle of gene delivery to synovium, chondrocytes and mesenchymal progenitor cells has been convincingly demonstrated. Following this, research efforts have begun to move to functional studies. This involves the identification of appropriate gene or gene combinations, incorporation of these cDNAs into appropriate vectors and delivery to specific target cells within the proper biological context to achieve a meaningful therapeutic response. Methods currently being explored range from those as simple as direct delivery of a vector to a cartilage defect, to synthesis of cartilaginous implants through gene-enhanced tissue engineering. Data from recent efficacy studies provide optimism that gene delivery can be harnessed to guide biological processes toward both accelerated and improved articular cartilage repair.