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。     

Persistence of fluorescent nanoparticle‐labelled bone marrow mesenchymal stem cells in vitro and after intra‐articular injection

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra‐articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD‐labelled MSCs [QD‐MSCs]) in vivo after intra‐articular injection into normal and osteoarthritic joints. One week after injection of QD‐MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD‐MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD‐MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD‐MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle‐injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD‐MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD‐MSCs can be visualized in histological sections 1 week after intra‐articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long‐term tracking of MSCs, especially under proliferative conditions.     

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1

The idea of rescuing the body self‐repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re‐entry in the cell cycle of confluent, growth‐arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up‐regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short‐ and long‐term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an “in vitro” tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia‐inducible factor (HIF‐1) alpha increase, its nuclear relocation and the binding to HIF‐1 responsive elements. These events were possibly related to the cell proliferation because the HIF‐1 inhibitor acriflavine inhibited HIF‐1 binding to HIF‐1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage.     

PGA‐associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non‐articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell‐cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid‐ (PGA) associated porcine heterotopic mono‐ and cocultures were assessed for cell vitality, types II, I, and total collagen‐, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1‐integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species‐dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan‐rich hyaline‐like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time‐dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair. Copyright © 2011 John Wiley & Sons, Ltd.     

Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage‐generating cells. Previous studies in static culture have shown that hACs co‐cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co‐culture of hMSCs and OA hACs under serum‐free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125‐ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75‐fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co‐culture as a 2D monolayer in static culture flasks, bioreactor co‐culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum‐free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell‐based cartilage repair.     

Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes

A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co‐culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non‐diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co‐cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co‐culture with cryopreserved passaged cells for functional analysis. The co‐culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline‐like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co‐culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I)

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2+/-22.3 ng/10(7) cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.     

人胚关节软骨细胞移植修复关节软骨缺损的免疫学研究

目的 通过非协调性异种移植，从免疫学角度探讨将人胚关节软骨细胞作为同种异体移植材料用于关节软骨损伤修复的可能性。方法 分离培养人胚关节软骨细胞，移植修复家兔膝关节软骨缺损，于术后2、4、8、12、20周通过淋巴细胞转化试验、细胞因子水平监测及循环中抗供者抗体的检测进行免疫学监测，同时设胶原对照组和空白对照组。结果 综合分析免疫学检测结果显示，采用人胚关节软骨细胞异种移植修复关节软骨缺损未见明显的宿主排斥移植物反应发生无明显。结论 本研究为人胚关节软骨细胞应用于同种异体移植修复关节软骨缺损提供了免疫学佐证。     

异种关节软骨与MSCs体外共同培养的实验研究

目的提供一种新的种子细胞获得方法,解决软骨缺损修复过程中软骨数量不足的困难。方法人骨髓间充质干细胞体外扩增诱导分化后与兔的关节软骨细胞按不同比例共同培养利用real-time PCR技术分析细胞表型的表达情况。结果实验组2种细胞共培养4周后,混合培养显示正反馈调节软骨细胞的增殖,软骨细胞外基质、SOX9、Ⅱ型胶原基因表达增加,软骨细胞生成明显增加。结论间充质干细胞以旁分泌或自分泌的方式调节间充质干细胞向软骨细胞的转化。     

Design,construction, and biological testing of an implantable porous trilayer scaffold for repairing osteoarthritic cartilage

Various tissue engineering systems for cartilage repair have been designed and tested over the past two decades, leading to the development of many promising cartilage grafts. However, no one has yet succeeded in devising an optimal system to restore damaged articular cartilage. Here, the design, assembly, and biological testing of a porous, chitosan/collagen‐based scaffold as an implant to repair damaged articular cartilage is reported. Its gradient composition and trilayer structure mimic variations in natural cartilage tissue. One of its layers includes hydroxyapatite, a bioactive component that facilitates the integration of growing tissue on local bone in the target area after scaffold implantation. The scaffold was evaluated for surface morphology; rheological performance (storage, loss, complex, and time‐relaxation moduli at 1 kHz); physiological stability; in vitro activity and cytotoxicity (on a human chondrocyte C28 cell line); and in vivo performance (tissue growth and biodegradability), in a murine model of osteoarthritis. The scaffold was shown to be mechanically resistant and noncytotoxic, favored tissue growth in vivo, and remained stable for 35 days postimplantation in mice. These encouraging results highlight the potential of this porous chitosan/collagen scaffold for clinical applications in cartilage tissue engineering.     

Low‐oxygen conditions promote synergistic increases in chondrogenesis during co‐culture of human osteoarthritic stem cells and chondrocytes

There has been increased interest in co‐cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion, and a diminished capacity to synthesise cartilage‐specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture‐expanded chondrocytes for cartilage repair applications; however, the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture‐expanded human chondrocytes with infrapatellar fat pad–derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF‐β3 only or TGF‐β3 and BMP‐7) conditions. Both co‐cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co‐cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O₂. Additionally, collagen type I synthesis was suppressed in co‐cultures maintained at low‐oxygen conditions. This study demonstrates that a co‐culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering.     

聚乙酸-聚乙醇酸共聚物复合同种异体细胞修复软骨缺损

背景:以自体骨髓间充质干细胞和软骨细胞作为种子细胞修复软骨缺损可达到理想效果,但存在细胞数量不足及二次创伤等问题。目的:观察同种异体骨髓间充质干细胞和软骨细胞与聚乙酸-聚乙醇酸共聚物复合修复关节软骨的可行性。方法:将15只新西兰大白兔随机分为实验组、对照组、空白组,制作关节软骨缺损模型,分别于骨缺损处植入同种异体骨髓间充质干细胞和同种异体软骨细胞与聚乙酸-聚乙醇酸共聚物复合体、自体骨髓间充质干细胞和自体软骨细胞与聚乙酸-聚乙醇酸共聚物复合体、聚乙酸-聚乙醇酸共聚物材料。结果与结论:术后12周苏木精-伊红及Masson三色染色显示,实验组软骨缺损处可见软骨细胞,呈圆形或多角形,柱状排列,软骨陷窝形成明显,可见大量细胞外基质沉积,修复组织显示出透明软骨样,与周围软骨结合好,与底层骨结合紧密;对照组与实验组无明显差别;空白组软骨缺损处可见纤维样细胞。说明同种异体骨髓间充质干细胞和软骨细胞与聚乙酸-聚乙醇酸共聚物复合可修复关节软骨缺损。     

应用生物蛋白胶与胚胎软骨细胞混合移植修复兔膝关节实验性软骨缺损区

背景应用异体软骨细胞移植修复关节软骨缺损的研究较多,但其可引起免疫反应.由于胚胎细胞具有较低的抗原性和较强的增殖能力,期望其能为组织工程研究与应用开辟新的载体材料.目的采用兔胚胎软骨细胞培养移植修复关节软骨缺损,并对其组织形态学进行观察.设计随机分组观察对比实验.单位广西医科大学组织胚胎学试验室.材料孕4周新西兰大白兔1只;成年新西兰大白兔24只,雌雄不限,体质量2～2.5 kg .方法实验于2000-12/2002-06在广西医科大学组织胚胎学试验室完成.在成兔股骨内侧髁作关节软骨缺损模型,实验组采用生物蛋白胶与胚胎软骨细胞相混后,植入兔膝关节实验性软骨缺损区,对照组不做任何处理或单纯用生物蛋白胶移植修补,分别于术后4,8,12周观察关节软骨缺损修复的情况,并按改良Pineda方法对其进行组织学评分,标准为细胞形态、基质染色、表面平整、软骨厚度、宿主结合5项,0分表示正常,分值越高表示改变越严重.主要观察指标①兔膝关节标本大体观察结果.②兔膝关节标本组织学观察结果.③关节软骨组织学半定量计分结果.④关节软骨缺损修复区进行疗效评价结果.结果24只兔均进入结果分析.①兔膝关节标本大体观察结果胚胎软骨细胞+纤维蛋白封闭剂组缺损区颜色接近正常软骨,质韧弹性好,与周围软骨界限消失;纤维蛋白封闭剂组与未处理组始终未愈合,但创面略小,有白色纤维组织充填.②兔膝关节标本组织学观察结果胚胎软骨细胞+纤维蛋白封闭剂组优势组织为透明样软骨,深方为骨组织,表面略凸或平整,基质着色正常,与周围软骨完全结合分界不清,组织中未发现淋巴细胞浸润;纤维蛋白封闭剂组与未处理组为纤维组织,部分有残留明显的凹陷性瘢痕,与周围组织接合或部分接合.③关节软骨组织学半定量计分结果按改良自Pineda方法,12周胚胎软骨细胞+纤维蛋白封闭剂组评分显著低于纤维蛋白封闭剂组和未处理组[(0.50±0.76),(7.88±1.13),(8.13±1.36)分,P＜0.05],而单纯用纤维蛋白封闭剂修补组在4周与未处理组比较有显著性差异(P＜0.05),在8,12周则无差异.④关节软骨缺损修复区进行疗效评价结果12周时胚胎软骨细胞+纤维蛋白封闭剂组完全修复8膝,纤维蛋白封闭剂组不完全修复1膝,未修复7膝,未处理组未修复8膝.结论胚胎软骨细胞移植组所产生的修复组织接近正常软骨组织,明显优于纤维蛋白封闭剂组和未处理组,该方法作为关节软骨缺损修复是可行的.     

The effects of using vitrified chondrocyte sheets on pain alleviation and articular cartilage repair

T he effect of using vitrified–thawed chondrocyte sheets on articular cartilage repair was examined because the methods for storing chondrocyte sheets are essential for allogeneic chondrocyte sheet transplantation. Six Japanese white rabbits were used as sources of articular chondrocytes and synovial cells. Chondrocytes were harvested from the femur, and synovial cells were harvested from inside the knee joints. After coculture of the chondrocytes with synovial cells, triple‐layered chondrocyte sheets were fabricated. Eighteen rabbits were used, with six rabbits in each of three groups: osteochondral defect only (control, group A); chondrocyte sheets (group B); and vitrified–thawed chondrocyte sheets (group C). An osteochondral defect was created on the femur. After transplantation, the weight distribution ratio of the undamaged and damaged limbs was measured as a pain‐alleviating effect. The rabbits were euthanized at 12 weeks, and the transplanted tissues were evaluated for histology (Safranin O staining and immunostaining) using the International Cartilage Repair Society grading system. For both evaluations, significant differences were observed between groups A and B, and between groups A and C (p < 0.05). No significant differences were observed between groups B and C. Thus, pain‐alleviating effects and tissue repair were achieved using vitrified–thawed chondrocyte sheets. Copyright © 2017 John Wiley & Sons, Ltd.     

骨髓间充质干细胞与同种异体脱钙骨基质组合生长因子诱导向软骨细胞分化

背景：用组织工程学方法高质量修复关节软骨缺损并达到很好的远期疗效目前尚无定论。鉴于此,课题组提出＂同种异体骨髓间充质干细胞关节腔内定向培养组织工程软骨＂的实验设技。目的：同种异体脱钙骨基质组合转化生长因子β1和胰岛素样生长因子Ⅰ诱导骨髓间充质干细胞向软骨分化的能力,同时探索其在关节腔内培养促进向关节软骨定向分化的方法。方法：分离兔骨髓间充质干细胞并行体外培养,分为两组：实验组DMEM培养液中加入转化生长因子β1和胰岛素样生长因子Ⅰ,对照组中未加入诱导因子。比较两组细胞的增殖情况和成软骨分化情况。制备同种异体脱钙骨基质支架材料,实验组骨髓间充质干细胞负载到同种异体脱钙骨基质中,构建组织工程软骨复合体,取另2只兔的腰背筋膜包裹后缝合固定到30只兔膝关节腔内行腔内培养。分别于植入后4,8,12周各取10只标本进行组织学切片观察及Ⅱ型胶原免疫组织化学观测,并对结果进行分析。结果与结论：实验组中骨髓间充质干细胞集落形成效率明显高于对照组（u=3.326,P〈0.01）。实验组细胞爬片做Ⅱ型胶原免疫组化检测呈阳性,对照组未见阳性细胞。组织工程复合体在腔内培养12周后,苏木精-伊红染色见大量软骨细胞增生,胞核染色呈蓝色;甲苯胺蓝染色见软骨细胞成串排列,大量软骨陷窝形成,周围大量基质包绕;Ⅱ型胶原免疫组织化学反应见细胞外基质中出现大量棕黄色颗粒,Ⅱ型胶原染色强阳性。提示转化生长因子β1和胰岛素样生长因子Ⅰ可显著促进骨髓间充质干细胞增殖和成软骨分化,骨髓间充质干细胞与同种异体脱钙骨基质结合后可在关节腔内成功培养出组织工程软骨。同种异体脱钙骨基质符合组织工程软骨支架材料的基本要求。     

兔关节软骨接受体外冲击波治疗后细胞增殖及Ⅱ型胶原的表达

背景：关节软骨损伤后自身修复能力有限,体外冲击波可能提供一种高质量的修复关节软骨损伤并达到很好远期疗效的方法。目的：探讨体外冲击波对兔关节软骨细胞增殖及Ⅱ型胶原蛋白表达的影响。方法：酶消化法获得正常兔膝关节软骨细胞,培养并传代,实验A、B、C组分别用0.5×105,1.5×105,2.5×105Pa等3种强度能量体外冲击波干预,空白对照组无任何干预措施。结果与结论：细胞生长曲线在第6天实验B组显著高于其他各组（P〈0.05）;ELISA法检测实验B组Ⅱ型胶原A值与其他各组相比,显著升高（P〈0.05）;RT-PCR法检测实验B组Ⅱ型胶原表达明显增强,与其他各组相比差异有显著性意义（P〈0.05）。提示适当能量强度及频次的体外冲击波刺激,能够明显促进软骨细胞的增殖及Ⅱ型胶原表达。     

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid‐based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage‐specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Activated platelet‐rich plasma improves cartilage regeneration using adipose stem cells encapsulated in a 3D alginate scaffold

In the current study, the effect of superimposing platelet‐rich plasma (PRP) on different culture mediums in a three‐dimensional alginate scaffold encapsulated with adipose‐derived mesenchymal stem cells for cartilage tissue repair is reported. The three‐dimensional alginate scaffolds with co‐administration of PRP and/or chondrogenic supplements had a significant effect on the differentiation of adipose mesenchymal stem cells into mature cartilage, as assessed by an evaluation of the expression of cartilage‐related markers of Sox9, collagen II, aggrecan and collagen, and glycosaminoglycan assays. For in vivo studies, following induction of osteochondral lesion in a rabbit model, a high degree of tissue regeneration in the alginate plus cell group (treated with PRP plus chondrogenic medium) compared with other groups of cell‐free alginate and untreated groups (control) were observed. After 8 weeks, in the alginate plus cell group, functional chondrocytes were observed, which produced immature matrix, and by 16 weeks, the matrix and hyaline‐like cartilage became completely homogeneous and integrated with the natural surrounding cartilage in the defect site. Similar effect was also observed in the subchondral bone. The cell‐free scaffolds formed fibrocartilage tissue, and the untreated group did not form a continuous cartilage over the defect by 16 weeks.     

Characterization of costal cartilage and its suitability as a cell source for articular cartilage tissue engineering

Costal cartilage is a promising donor source of chondrocytes to alleviate cell scarcity in articular cartilage tissue engineering. Limited knowledge exists, however, on costal cartilage characteristics. This study describes the characterization of costal cartilage and articular cartilage properties and compares neocartilage engineered with costal chondrocytes to native articular cartilage, all within a sheep model. Specifically, we (a) quantitatively characterized the properties of costal cartilage in comparison to patellofemoral articular cartilage, and (b) evaluated the quality of neocartilage derived from costal chondrocytes for potential use in articular cartilage regeneration. Ovine costal and articular cartilages from various topographical locations were characterized mechanically, biochemically, and histologically. Costal cartilage was stiffer in compression but softer and weaker in tension than articular cartilage. These differences were attributed to high amounts of glycosaminoglycans and mineralization and a low amount of collagen in costal cartilage. Compared to articular cartilage, costal cartilage was more densely populated with chondrocytes, rendering it an excellent chondrocyte source. In terms of tissue engineering, using the self‐assembling process, costal chondrocytes formed articular cartilage‐like neocartilage. Quantitatively compared via a functionality index, neocartilage achieved 55% of the medial condyle cartilage mechanical and biochemical properties. This characterization study highlighted the differences between costal and articular cartilages in native forms and demonstrated that costal cartilage is a valuable source of chondrocytes suitable for articular cartilage regeneration strategies.     

Effect of the small compound TD‐198946 on glycosaminoglycan synthesis and transforming growth factor β3‐associated chondrogenesis of human synovium‐derived stem cells in vitro

As an alternative to chondrocytes‐based cartilage repair, stem cell‐based therapies have been investigated. Specifically, human synovium‐derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD‐198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD‐198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD‐198946 were examined in chondrogenic cultures of hSSC‐derived cell pellets. The effect of TD‐198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD‐198946 dose‐dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor β3 (TGFβ3), TD‐198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD‐198946 attenuated TGFβ3‐associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD‐198946 as judged by safranin O staining. Thus, TD‐198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD‐198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFβ3 + 0.25 nM TD‐198946. These studies have demonstrated that TD‐198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell‐based cartilage therapy.