In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells,employing a two‐chambered co‐culture well design

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk–RADA peptide scaffold in a specially designed two‐chambered co‐culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two‐chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co‐culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage‐like and subchondral bone‐like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single‐step approach that highlights the feasibility of rabbit BMSCs as a single‐cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Multiphasic collagen fibre–PLA composites seeded with human mesenchymal stem cells for osteochondral defect repair: an in vitro study

A promising approach for the repair of osteochondral defects is the use of a scaffold with a well‐defined cartilage–bone interface. In this study, we used a multiphasic composite scaffold with an upper collagen I fibre layer for articular cartilage repair, separated by a hydrophobic interface from a lower polylactic acid (PLA) part for bone repair. Focusing initially on the engineering of cartilage, the upper layer was seeded with human mesenchymal stem cells (hMSCs) suspended in a collagen I hydrogel for homogeneous cell distribution. The constructs were cultured in a defined chondrogenic differentiation medium supplemented with 10 ng/ml transforming growth factor‐β1 (TGFβ1) or in DMEM with 10% fetal bovine serum as a control. After 3 weeks a slight contraction of the collagen I fibre layer was seen in the TGFβ1‐treated group. Furthermore, a homogeneous cell distribution and chondrogenic differentiation was achieved in the upper third of the collagen I fibre layer. In the TGFβ1‐treated group cells showed a chondrocyte‐like appearance and were surrounded by a proteoglycan and collagen type II‐rich extracellular matrix. Also, a high deposition of glycosaminoglycans could be measured in this group and RT–PCR analyses confirmed the induction of chondrogenesis, with the expression of cartilage‐specific marker genes, such as aggrecan and collagen types II and X. This multiphasic composite scaffold with the cartilage layer on top might be a promising construct for the repair of osteochondral defects. Copyright © 2009 John Wiley & Sons, Ltd.     

动物源性骨软骨支架复合骨髓间充质干细胞/软骨细胞修复兔膝关节骨软骨复合缺损

背景:以往支架材料修复骨软骨的实验大都存在骨软骨耦合界面修复不良的情况.目的:观察骨髓间充质干细胞/软骨细胞复合动物源性骨软骨支架修复兔膝关节骨软骨复合缺损的可行性.方法:将新西兰大白兔随机抽签分为实验组、对照组、空白组,制作单侧膝关节骨软骨复合缺损后,实验组于骨缺损处植入自体骨髓间充质干细胞/诱导分化的软骨细胞与同种异体动物源性骨软骨复合支架,对照组于骨缺损处植入同种异体动物源性骨软骨支架、空白组未植入任何材料.术后4,8,12周行大体观察、苏木精-伊红染色、甲苯胺蓝染色.结果与结论:实验组大体观察见复合缺损区完全修复,局部无凹陷,新生组织和周围组织融合,苏木精-伊红染色和甲苯胺蓝染色见软骨缺损区由新生的透明软骨样组织修复,细胞柱状排列,极性好,软骨陷窝明显,骨缺损区由骨样组织修复,新生软骨和软骨下骨以及宿主骨界面耦合良好,甲苯胺蓝染色阳性率和组织学评分优于对照组、空白组(P < 0.05).说明诱导分化的自体软骨细胞和骨髓间充质干细胞共培养复合动物源性骨软骨支架所构建的细胞-支架复合体能成功修复兔膝关节软骨和软骨下骨的复合缺损,是一种理想的骨软骨复合缺损修复方法.     

动物源性骨软骨支架复合骨髓间充质干细胞/软骨细胞修复兔膝关节骨软骨复合缺损（英文）

背景：以往支架材料修复骨软骨的实验大都存在骨软骨耦合界面修复不良的情况。目的：观察骨髓间充质干细胞/软骨细胞复合动物源性骨软骨支架修复兔膝关节骨软骨复合缺损的可行性。方法：将新西兰大白兔随机抽签分为实验组、对照组、空白组,制作单侧膝关节骨软骨复合缺损后,实验组于骨缺损处植入自体骨髓间充质干细胞/诱导分化的软骨细胞与同种异体动物源性骨软骨复合支架,对照组于骨缺损处植入同种异体动物源性骨软骨支架、空白组未植入任何材料。术后4,8,12周行大体观察、苏木精-伊红染色、甲苯胺蓝染色。结果与结论：实验组大体观察见复合缺损区完全修复,局部无凹陷,新生组织和周围组织融合,苏木精-伊红染色和甲苯胺蓝染色见软骨缺损区由新生的透明软骨样组织修复,细胞柱状排列,极性好,软骨陷窝明显,骨缺损区由骨样组织修复,新生软骨和软骨下骨以及宿主骨界面耦合良好,甲苯胺蓝染色阳性率和组织学评分优于对照组、空白组（P〈0.05）。说明诱导分化的自体软骨细胞和骨髓间充质干细胞共培养复合动物源性骨软骨支架所构建的细胞-支架复合体能成功修复兔膝关节软骨和软骨下骨的复合缺损,是一种理想的骨软骨复合缺损修复方法。     

体外构建组织工程骨-软骨复合组织

背景：设计一体化、具有过渡结构的双层支架材料,复合软骨细胞、骨髓间充质细胞,有利于新生的骨与软骨组织之间形成良好界面。目的：模仿自然骨一软骨基质构建复合支架,以软骨细胞和骨髓间充质干细胞为种子细胞,体外观察复合组织的成软骨及成骨能力。方法：制备明胶一硫酸软骨素一透明质酸及明胶一陶瓷化骨多孔复合支架,构建自然骨一软骨基质复合支架,复合兔软骨细胞与骨髓间充质干细胞,分未成骨诱导与成骨诱导两组培养,并进行MTT、糖胺多糖含量、碱性磷酸酶活性检测,以及苏木精一伊红染色检测。结果与结论：未成骨诱导与成骨诱导两组骨髓间充质干细胞增殖及糖胺多糖含量差异无显著性意义。未成骨诱导组碱性磷酸酶活性缓慢上升,成骨诱导组诱导后碱性磷酸酶活性迅速上升,14d时达到稳定状态。两组苏木精一伊红染色结果无明显区别,均已形成含有双层组织的类似骨一软骨样组织,其间可见未降解支架形态,但由于基质形成不完善及支架未完全降解,此种结构不成熟,细胞分布不均匀,支架内部可见散在无细胞区域。证实采用两种细胞与双层结构的支架经体外分层复合能够形成组织工程骨软骨复合组织。     

Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits

The purpose of this study was to track mesenchymal stem cells (MSCs) labelled with internalizing quantum dots (i‐QDs) in the reparative tissues, following the allogeneic transplantation of three‐dimensional (3D) cartilaginous aggregates into the osteochondral defects of rabbits. QDs were conjugated with a unique internalizing antibody against a heat shock protein‐70 (hsp70) family stress chaperone, mortalin, which is upregulated and expressed on the surface of dividing cells. The i‐QDs were added to the culture medium for 24 h. Scaffold‐free cartilaginous aggregates formed from i‐QD‐labelled MSCs (i‐MSCs), using a 3D culture system with chondrogenic supplements for 1 week, were transplanted into osteochondral defects of rabbits. At 4, 8 and 26 weeks after the transplantation, the reparative tissues were evaluated macroscopically, histologically and fluoroscopically. At as early as 4 weeks, the defects were covered with a white tissue resembling articular cartilage. In histological appearance, the reparative tissues resembled hyaline cartilage on safranin‐O staining throughout the 26 weeks. In the deeper portion, subchondral bone and bone marrow were well remodelled. On fluoroscopic evaluation, QDs were tracked mainly in bone marrow stromata, with some signals detected in cartilage and the subchondral bone layer. We showed that the labelling of rabbit MSCs with anti‐mortalin antibody‐conjugated i‐QDs is a tolerable procedure and provides a stable fluorescence signal during the cartilage repair process for up to 26 weeks after transplantation. The results suggest that i‐MSCs did not inhibit, and indeed contributed to, the regeneration of osteochondral defects. Copyright © 2010 John Wiley & Sons, Ltd.     

Chondrogenic,hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three‐dimensionally woven scaffolds

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three‐dimensionally woven poly(ε‐caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations—PCL alone, PCL‐bone, “point‐of‐care” seeded MSC‐PCL‐bone, and chondrogenically precultured Ch‐MSC‐PCL‐bone constructs—were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch‐MSC‐PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula‐like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.     

Micro‐aggregates do not influence bone marrow stromal cell chondrogenesis

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC‐based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro‐aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ₃ added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro‐aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro‐aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro‐aggregates. Copyright © 2014 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.     

A Col I and BCP ceramic bi-layer scaffold implant promotes regeneration in osteochondral defects

Osteochondral defects occur in the superficial cartilage region, intermediate calcified cartilage, and subchondral bone. Due to the limited regenerative capacity and complex zonal structure, it is critically difficult to develop strategies for osteochondral defect repair that could meet clinical requirements. In this study, type I collagen (Col I) and BCP ceramics were used to fabricate a new bi-layer scaffold for regeneration in osteochondral defects. The in vitro studies showed that the bi-layer scaffold provided special functions for cell migration, proliferation and secretion due to the layered scaffold structure. The in vivo results demonstrated that the bi-layered scaffold could effectively promote the regeneration of both the cartilage and the subchondral bone, and the newly formed cartilage layer, with a similar structure and thickness to the natural cartilaginous layer, could seamlessly integrate with the surrounding natural cartilage and regenerate an interface layer to mimic the native osteochondral structure.

A new bi-layer scaffold composed of Col I and BCP ceramic was prepared to regenerate osteochondral defect. The result demonstrated the bi-layer scaffold could effectively promote the regeneration of both the cartilage and the subchondral bone layer.     

Dihydromyricetin enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells in vitro partially via the activation of Wnt/β‐catenin signaling pathway

Substantial evidence has demonstrated that the decreased osteogenic differentiation of bone mesenchymal stem cells (BMSCs) is closely related to bone metabolic diseases. Thus, it is very important to develop several potentially useful therapeutic agents to enhance BMSC osteogenesis. Flavonoids show promise in enhancing bone mass. Dihydromyricetin (DMY), a type of flavonoid, has not yet been investigated regarding its effects on BMSC osteogenesis. To investigate the effects of DMY on osteogenesis, human BMSCs were induced with or without DMY. We found that DMY (0.1–50 μm ) exhibited no cytotoxic effect on proliferation, but increased alkaline phosphatase activity, osteoblast‐specific gene expression, and mineral deposition. It also enhanced active β‐catenin expression and reduced dickkopf‐1(DKK1) and sclerostin expression. The Wnt/β‐catenin signaling pathway inhibitor (DKK1 and β‐catenin‐specific siRNA) decreased the enhanced bone mineral formation caused by DMY. Taken together, these findings reveal that DMY enhances osteogenic differentiation of human BMSCs partly through Wnt/β‐catenin in vitro.     

Design and characterization of a tissue‐engineered bilayer scaffold for osteochondral tissue repair

Treatment of full‐thickness cartilage defects relies on osteochondral bilayer grafts, which mimic the microenvironment and structure of the two affected tissues: articular cartilage and subchondral bone. However, the integrity and stability of the grafts are hampered by the presence of a weak interphase, generated by the layering processes of scaffold manufacturing. We describe here the design and development of a bilayer monolithic osteochondral graft, avoiding delamination of the two distinct layers but preserving the cues for selective generation of cartilage and bone. A highly porous polycaprolactone‐based graft was obtained by combining solvent casting/particulate leaching techniques. Pore structure and interconnections were designed to favour in vivo vascularization only at the bony layer. Hydroxyapatite granules were added as bioactive signals at the site of bone regeneration. Unconfined compressive tests displayed optimal elastic properties and low residual deformation of the graft after unloading (< 3%). The structural integrity of the graft was successfully validated by tension fracture tests, revealing high resistance to delamination, since fractures were never displayed at the interface of the layers (n = 8). Ectopic implantation of grafts in nude mice, after seeding with bovine trabecular bone‐derived mesenchymal stem cells and bovine articular chondrocytes, resulted in thick areas of mature bone surrounding ceramic granules within the bony layer, and a cartilaginous alcianophilic matrix in the chondral layer. Vascularization was mostly observed in the bony layer, with a statistically significant higher blood vessel density and mean area. Thus, the easily generated osteochondral scaffolds, since they are mechanically and biologically functional, are suitable for tissue‐engineering applications for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

完全脱钙骨基质与骨髓间充质干细胞体外诱导构建组织工程纤维软骨

背景：含有骨形态发生蛋白的完全脱钙骨基质可使骨髓间充质干细胞向软骨细胞分化并维持其软骨细胞的特性,在软骨发生过程中发挥重要作用。目的：将骨髓间充质干细胞与完全脱钙骨基质在体外诱导培养成纤维软骨组织以及观察培养体系与支架材料对细胞凋亡的影响。方法：采用全骨髓培养法分离培养猪骨髓间充质干细胞,取第1代骨髓间充质干细胞均匀接种到完全脱钙骨基质上,以含有转化生长因子β1、胰岛素样生长因子、地塞米松、抗坏血酸及体积分数10%FBS的高糖DMEM诱导培养液培养作为实验组,将单层诱导培养、单层基础培养的骨髓间充质干细胞、空白完全脱钙骨基质作为对照,于培养第1,2,3,4周进行大体形态观察以及Ⅰ、Ⅱ型胶原及聚集蛋白聚糖的RT-PCR定性检测;培养4周进行细胞凋亡检测。结果与结论：实验组可检测到Ⅰ型胶原的持续表达,Ⅱ型胶原及聚集蛋白聚糖表达逐渐增加。单层诱导培养组可检测到Ⅱ型胶原、聚集蛋白聚糖的表达及Ⅰ型胶原的持续表达,但表达量远低于实验组;实验组细胞凋亡率高于单层诱导培养组、单层基础培养组。说明转化生长因子β1、胰岛素样生长因子1体外诱导培养的骨髓间充质干细胞-完全脱钙骨基质可以表达特异性软骨基质,三维环境培养细胞外基质的表达量明显高于单层诱导培养;静态培养系统对三维培养的细胞凋亡有影响。     

Repair potential of nonsurgically delivered induced pluripotent stem cell‐derived chondrocytes in a rat osteochondral defect model

Human induced pluripotent stem cells (hiPSCs) are thought to be an alternative cell source for future regenerative medicine. hiPSCs may allow unlimited production of cell types that have low turnover rates and are difficult to obtain such as autologous chondrocytes. In this study, we generated hiPSC‐derived chondrogenic pellets, and chondrocytes were isolated. To confirm the curative effects, chondrogenic pellets and isolated chondrocytes were transplanted into rat joints with osteochondral defects. Isolated hiPSC‐derived chondrocytes were delivered in the defect by a single intra‐articular injection. The generated hiPSC‐derived chondrogenic pellets had increased chondrogenic marker expression and accumulated extracellular matrix proteins. Chondrocytes were successfully isolated from the pellets. Alcian blue staining and collagen type II were detected in the cells. Chondrogenic marker expression was also increased in the isolated cells. Transplanted chondrogenic pellets and chondrocytes both had curative effects in the osteochondral defect rat model. Detection of human proteins in the joints proved that the cells were successfully delivered into the defect. Chondrogenic pellets or chondrocytes generated from hiPSCs have potential as regenerative medicine for cartilage recovery or regeneration. Chondrocytes isolated from hiPSC‐derived chondrogenic pellets had curative effects in damaged cartilage. Injectable hiPSC‐derived chondrocytes show the possibility of noninvasive delivery of regenerative medicine for cartilage recovery.     

自体骨髓基质干细胞及可注射藻酸钙凝胶联合Mosaicplasly技术修复山羊膝关节股骨头大面积缺损

背景:目前修复软骨缺损的方法都存在修复组织数量不足,生物力学性能不佳,整合不良及供区并发症等缺陷.对于大面积的骨软骨复合缺损单独应用一种方法尚显不足.目的:观察组织工程方法复合Mosaicplasty技术用于修复大面积骨软骨缺损的效果.设计、时间及地点:随机对照动物实验,于2009-01/09在青岛大学医学院中心实验室完成.材料:体外培养并扩增健康成年雄性山羊的骨髓基质干细胞,收集约3×10~7细胞,加入1 mL藻酸钠溶液重悬形成骨髓基质干细胞一藻酸钙凝胶材料.方法:12只山羊用于制各膝关节股骨髁大面积骨软骨缺损模型,骨髓基质干细胞-Mosaicplasty组使用自制Mosaicplasty器械,植入直径2 mm骨软骨柱镶嵌充填缺损,以自体骨髓基质干细胞复合藻酸钙凝胶填充残余缺损和部分供区.Mosaicplasty组单纯用Mosaicplasty修复骨软骨缺损.对照组单纯制造缺损不修复.主要观察指标:①大体观察:术后4,8,16周分别切开关节观察修复效果.②组织学检查:术后16周取修复组织标本,行苏木精-伊红染色、甲苯胺蓝染色光镜下观察.③电镜观察:取16周修复组织行透射电镜检查.结果;术后16周时骨髓基质干细胞-Mosaicplasty组移植物固定牢固,关节面平滑,移植物间界限消失,新生软骨组织类似于正常软骨,4-16周修复效果逐渐改善,优于其他各组.光镜观察细胞-凝胶新生软骨组织与移植软骨结合紧密,新生软骨细胞排列规整,细胞外基质分布均一.对照组无明显修复.透射电镜观察发现修复新生组织中细胞形态类似软骨细胞,细胞存在于排列紧密的纤维网格中,基质丰富.结论:使用自体骨髓基质干细胞-藻酸钙凝胶材料复合Mosaicplasty技术可促进骨软骨整合,改善其修复效果.     

Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Regeneration of osteochondral defects in vivo by a cell‐free cylindrical poly(lactide‐co‐glycolide) scaffold with a radially oriented microstructure

A scaffold with an oriented porous architecture to facilitate cell infiltration and bioactive interflow between neo‐host tissues is of great importance for in situ inductive osteochondral regeneration. In this study, a poly(lactide‐co‐glycolide) (PLGA) scaffold with oriented pores in its radial direction was fabricated via unidirectional cooling of the PLGA solution in the radial direction, following with lyophilization. Micro‐computed tomography evaluation and scanning electron microscopy observation confirmed the radially oriented microtubular pores in the scaffold. The scaffold had porosity larger than 90% and a compressive modulus of 4 MPa in a dry state. Culture of bone marrow stem cells in vitro revealed faster migration and regular distribution of cells in the poly(lactide‐co‐glycolide) scaffold with oriented pores compared with the random PLGA scaffold. The cell‐free oriented macroporous PLGA scaffold was implanted into rabbit articular osteochondral defect in vivo for 12 weeks to evaluate its inductive tissue regeneration function. Histological analysis confirmed obvious tide mark formation and abundant chondrocytes distributed regularly with obvious lacunae in the cartilage layer. Safranin O‐fast green staining showed an obvious boundary between the two layers with distinct staining results, indicating the simultaneous regeneration of the cartilage and subchondral bone layers, which is not the case for the random poly(lactide‐co‐glycolide) scaffold after the same implantation in vivo. The oriented macroporous PLGA scaffold is a promising material for the in situ inductive osteochondral regeneration without the necessity of preseeding cells.