Repair of large osteochondritis dissecans lesions using a novel multilayered tissue engineered construct in an equine athlete

Osteochondral lesions resulting from osteochondritis dissecans are problematic to treat and present a significant challenge for clinicians. The aims of this study were to investigate the use of a scaffold‐assisted microfracture approach, employing a novel, multilayered, collagen‐based, osteochondral graft substitute in the treatment of severe osteochondritis dissecans of both lateral femoral trochlear ridges in an equine athlete, and to assess the potential of this novel scaffold to enhance repair of the osteochondral unit. A 15 month‐old female filly presented with large osteochondritis dissecans lesions involving both femoral lateral trochlear ridges. After routine arthroscopic debridement and microfracture of the subchondral bone, multilayered osteochondral defect repair scaffolds were implanted into the fragmentation beds in both left and right femoropatellar joints via mini‐arthrotomies. Exploratory arthroscopy 5 months postimplantation revealed smooth cartilaginous repair tissue, contiguous with the adjacent cartilage, covering the defect. At 22‐month follow up, the filly had no signs of lameness and was exercising at her intended level. Radiographically, although still slightly flattened, the femoral trochlear ridges were smooth, with no evidence of osteoarthritis. Ultrasonographically, the defects were filled with bone and covered with an overlying cartilaginous layer, with the trochlear ridge contour almost entirely restored. This report demonstrates the effective clinical use of this novel, multilayered, osteochondral defect repair scaffold in the treatment of osteochondritis dissecans of an equine athlete. The successful repair achieved here using this novel scaffold in an equine patient with large bilateral lesions shows the potential for clinical translation in the treatment of human patients presenting with osteochondral defects. Copyright © 2016 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.     

多孔丝素蛋白/羟基磷灰石复合脂肪间充质干细胞修复兔关节软骨及软骨下骨缺损

背景：丝素蛋白/羟基磷灰石是细胞立体培养的良好支架,是临床常用的骨缺损修复材料,具有良好的生物相容性。脂肪干细胞具有向骨及软骨细胞分化的潜能,适合骨软骨缺损修复。目的：观察转化生长因子β1和胰岛素样生长因子1联合成软骨诱导脂肪干细胞与丝素蛋白/羟基磷灰石复合后修复兔关节软骨及软骨下骨缺损的效果。方法：取新西兰大白兔56只,2只用于传代培养脂肪间充质干细胞,以3×109L-1浓度接种到丝素蛋白/羟基磷灰石。其余54只新西兰大白兔,在股骨髁间制备软骨缺损模型,随机分为细胞复合材料组、单纯材料组和空白对照组,细胞复合材料组植入复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石;单纯材料组植入丝素蛋白/羟基磷灰石;空白对照组不作任何植入。从大体、影像学、组织学观察比较缺损的修复情况。结果与结论：12周时大体观察、CT、磁共振和组织学检查细胞材料复合组软骨及软骨下骨缺损区完全被软骨组织修复,修复组织与周围软骨色泽相近,支架材料基本吸收,未见明显退变和白细胞浸润,所有标本均未见丝素蛋白残留。单纯材料组缺损区缩小、部分修复,且呈纤维软骨样修复。空白对照组缺损无明显修复。提示复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石修复兔关节软骨及软骨下骨缺损能力优于单纯丝素蛋白/羟基磷灰石材料。丝素蛋白/羟基磷灰石复合脂肪间充质干细胞可形成透明软骨修复动物膝关节全层软骨缺损,重建关节的解剖结构和功能,可作为新型骨软骨组织工程支架。     

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。     

Tissue‐engineered constructs: the effect of scaffold architecture in osteochondral repair

Cartilage has a poor regenerative capacity. Tissue‐engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55 wt% poly(ethylene oxide terephthalate) and 45 wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three‐dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3 weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3 weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post‐implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM‐treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. Copyright © 2012 John Wiley & Sons, Ltd.     

Optimization of photocrosslinked gelatin/hyaluronic acid hybrid scaffold for the repair of cartilage defect

There is no therapy currently available for fully repairing articular cartilage lesions. Our laboratory has recently developed a visible light‐activatable methacrylated gelatin (mGL) hydrogel, with the potential for cartilage regeneration. In this study, we further optimized mGL scaffolds by supplementing methacrylated hyaluronic acid (mHA), which has been shown to stimulate chondrogenesis via activation of critical cellular signalling pathways. We hypothesized that the introduction of an optimal ratio of mHA would enhance the biological properties of mGL scaffolds and augment chondrogenesis of human bone marrow‐derived mesenchymal stem cells (hBMSCs). To test this hypothesis, hybrid scaffolds consisting of mGL and mHA at different weight ratios were fabricated with hBMSCs encapsulated at 20 × 10⁶ cells/ml and maintained in a chondrogenesis‐promoting medium. The chondrogenenic differentiation of hBMSCs, within different scaffolds, was estimated after 8 weeks of culture. Our results showed that mGL/mHA at a 9:1 (%, w/v) ratio resulted in the lowest hBMSC hypertrophy and highest glycosaminoglycan production, with a slightly increased volume of the entire construct. The applicability of this optimally designed mGL/mHA hybrid scaffold for cartilage repair was then examined in vivo. A full‐thickness cylindrical osteochondral defect was surgically created in the rabbit femoral condyle, and a three‐dimensional cell–biomaterial construct was fabricated by in situ photocrosslinking to fully fill the lesion site. The results showed that implantation of the mGL/mHA (9:1) construct resulted in both cartilage and subchondral bone regeneration after 12 weeks, supporting its use as a promising scaffold for repair and resurfacing of articular cartilage defects, in the clinical setting.     

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.     

羟基丁酸-羟基辛酸一体化骨软骨支架的体外血管化

背景：前期实验构建的羟基丁酸-羟基辛酸共聚体一体化骨软骨支架具备良好的生物相容性、生物可降解性，并且降解产物无毒性。 目的：将兔肾微血管内皮细胞与羟基丁酸-羟基辛酸一体化骨软骨支架复合培养，观察支架骨层血管化效果。方法：运用溶剂浇铸-颗粒沥滤法，制备具有骨层/骨与软骨界面层/软骨层3层结构的羟基丁酸-羟基辛酸一体化骨软骨支架。将传代培养至第3代的兔肾微血管内皮细胞，接种到一体化骨软骨支架骨层支架上，MTT法检测细胞在支架上的增殖活性，10 d后苏木精-伊红染色及电镜观察细胞在支架内的生长状况。 结果与结论：一体化骨软骨支架外观具备明显的3层结构，各层之间连接紧密，骨层疏松多孔，各层支架孔隙均匀且相通，一体化支架孔隙率为78%。兔肾微血管内皮细胞在支架上分裂增殖良好，复合培养10 d后，细胞在骨层支架内呈立体生长，中间界面层内未发现细胞，苏木精-伊红染色可见细胞黏附生长于骨层支架孔隙间，细胞依附支架的多孔结构生长，形成管腔样结构，但细胞并未长入中间界面层。     

Knorpeldefekte

Introduction

Hyaline articular cartilage covers the articulating surfaces of diarthrodial joints. The specific cartilage cells (chondrocytes) only account for approximately 2?% of the overall tissue volume. The main constituents of the cartilaginous extracellular matrix are type II collagen, proteoglycans and a diversity of other proteins and glycoproteins. These components are characteristically arranged in a three-dimensional network, comparable to a fiber-reinforced, fluid-saturated and permeable composite.

Methods

Articular cartilage defects are classified as being either chondral, exclusively affecting the articular cartilage layer or osteochondral, which also involves the subchondral bone compartment. Chondral defects may be either partial thickness or full thickness when extending into the calcified cartilage layer. As the subchondral bone plate remains intact in chondral defects, such lesions are (sparsely) repopulated by cells originating from the synovial membrane. In osteochondral defects, the integrity of the subchondral bone plate is disrupted, allowing mesenchymal stem cells from the bone marrow cavity to support and participate in the cartilage repair process.

Conclusion

Such repair of the entire osteochondral unit is associated with specific changes of the subchondral bone; however, the cartilage repair tissue of both chondral and osteochondral defects remains structurally and biomechanically inferior to the original hyaline cartilage and degenerates over time, potentially inducing secondary osteoarthritis.

     

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.     

Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses

The present in vivo preliminary experiment is aimed at testing mechanical and biological behaviour of a new nano‐structured composite multilayer biomimetic scaffold for the treatment of chondral and osteochondral defects. The three‐dimensional biomimetic scaffold (Fin‐Ceramica Faenza S.p.A., Faenza—Italy) was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles, in two configurations, bi‐ and tri‐layered, to reproduce, respectively, chondral and osteochondral anatomy. Chondral defects (lateral condyle) and deep osteochondral defects (medial condyle) were made in the distal epiphysis of the third metacarpal bone of both forelimbs of two adult horses and treated respectively with the chondral and osteochondral grafts. Both animals were euthanised six months follow up. The images obtained at the second look arthroscopy evaluation, performed two months after surgery, demonstrated good filling of the chondral and osteo‐chondral defects without any inflammatory reaction around and inside the lesions. At the histological analysis the growth of trabecular bone in the osteochondral lesion was evident. Only in one case, the whole thickness of the osteochondral lesion was filled by fibrocartilaginous tissue. The formation of a tidemark line was evident at the interface with the newly formed bone. Newly formed fibrocartilaginous tissue was present in the area of the chondral defect. Initial alignment of the collagen fibres was recognisable with polarised light in both groups. The results of the present pilot study showed that this novel osteochondral and chondral scaffold may act as a suitable matrix to facilitate orderly regeneration of bone and hyaline‐like cartilage. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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 generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide‐based scaffold

Tissue engineering of a biological osteochondral multilayered construct with a cartilage‐interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold‐based co‐culture approach to generate tissue‐engineered osteochondral grafts with an interface. BMSC‐seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self‐assembling peptide and subsequently co‐cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage‐subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co‐cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP‐13 were found on both chondrogenic and osteogenic section edges after co‐culture. However, significant differences gene expression profile were found in distinct zones of the construct during co‐culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co‐culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs. Copyright © 2013 John Wiley & Sons, Ltd.     

羟基丁酸与羟基辛酸共聚物一体化骨软骨组织工程支架的制备及性能

背景:单层支架难以满足关节软骨损伤修复的要求,现提出骨软骨共同修复的一体化支架,以弥补了单一支架的部分缺陷。目的:以羟基丁酸与羟基辛酸共聚物为基础材料,羟基磷灰石等为复合材料研制一体化骨软骨组织工程支架,测试该支架的物理特性和细胞黏附性。方法:采用溶剂浇铸/颗粒沥滤法,以支架孔径、孔隙率、力学强度和细胞黏附生长率为检测指标,以羟基丁酸与羟基辛酸共聚物为连续相,通过改变致孔剂NaCl粒径和羟基磷灰石材料配比制备不同形态结构、力学强度和生物学功能的三层一体化骨软骨组织工程支架。结果与结论:致孔剂与支架材料的最佳质量配比分别为软骨层4.5/1,过渡层2.5/1,硬骨层3.5/1。扫描电镜观察显示支架的三层结构明显不同且紧密结合,其软骨层、过渡层、硬骨层的孔径分别为150~250μm,≤60μm,150~450μm;孔隙率检测结果依次为84%,60%,75%;力学强度测定依次为2.93,6.43,4.30MPa;支架对骨髓间充质干细胞无毒性,细胞黏附与生长状态良好。结果表明该一体化骨软骨组织工程支架具有仿生学特性,符合骨软骨组织工程支架的基本条件。     

Utilizing tissue‐engineered cartilage or BMNC‐PLGA composites to fill empty spaces during autologous osteochondral mosaicplasty in porcine knees

The potential empty spaces between cylindrical plugs remaining after autologous osteochondral mosaicplasty rely on fibrous repair, which may constrain the quality and integrity of the repair. Thus, the empty spaces should be repaired, and how to fill the empty spaces is still a problem. In the present study, a standardized full‐thickness defect (diameter, 6 mm) was created in the weight‐bearing area of each medial femoral condyle in both knees of 18 miniature pigs. The 36 knees were randomly assigned to four groups with nine in each group. The defects were initially repaired by autologous osteochondral mosaicplasty. Simultaneously, any empty spaces between the multiple plugs were filled with cell‐free poly(lactide‐co‐glycolide) (PLGA) scaffolds (the scaffold group), tissue‐engineered cartilage (the TE group) or bone marrow mononuclear cell (BMNC)–PLGA composites (the composite group). The empty spaces were left untreated as control (the control group). Six months after surgery, the repair results were assessed via macroscopic observation, histological evaluation, magnetic resonance imaging, biomechanical assessment and glycosaminoglycan content. The results demonstrated that mosaicplasty combined with the treatment of the empty spaces could improve cartilage regeneration. The filling of empty spaces by tissue‐engineered cartilage produced the best result in all the four groups. Meanwhile, utilizing BMNC–PLGA composites achieved a similar repair result. Considering the cost‐effective, time‐saving and convenient performance, the BMNC‐PLGA composite could be an alternative option to fill the empty spaces combined with mosaicplasty. Copyright © 2014 John Wiley & Sons, Ltd.     

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。