组织工程化软骨修复界面整合的体外模型

背景：随着组织工程学的发展,自体软骨细胞移植技术经常被用来修复软骨缺损,整合不良是导致修复失败的原因之一。许多体外模型被用来进行这方面的研究。 目的：建立一种组织工程化软骨修复界面整合的体外实验模型并评价其效果。 方法：制备猪体外软骨整合模型,获得21个软骨环,18只琼脂糖凝胶覆盖的软骨环设为琼脂糖凝胶组,剩余3个做无琼脂糖对照组,分别植入分离的软骨细胞,观察近期软骨环边界细胞漏出情况,分别在1,2,4周做切片、染色并行组织学观察,测量新生软骨平均面积并进行比较。 结果与结论：无琼脂糖对照组由于软骨细胞早期从软骨环底部漏出,未能在软骨环中形成软骨细胞聚集,所以未做后期处理,而琼脂糖凝胶组则未发生。琼脂糖凝胶组1,2,4周做切片并行固定后组织切片分别用苏木精-伊红染色、阿利新蓝、番红O、Ⅱ型胶原免疫组化染色,移植的软骨细胞在软骨环内不断增殖,并且产生细胞外基质。在第1,2周的孵育中,新生软骨的面积明显增大,到第4周时,面积也有进一步增加,但是第2-4周的面积增加,差异无显著性意义（P〉0.05）。模型成功模拟了自体软骨细胞移植修复关节软骨缺损的体外整合过程,未来可应用于软骨整合及软骨组织工程的机制研究。     

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.     

Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs

When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.     

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.     

Translating the application of transforming growth factor‐β1, chondroitinase‐ABC,and lysyl oxidase‐like 2 for mechanically robust tissue‐engineered human neocartilage

Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor‐β1 [T]), a biophysical agent (chondroitinase‐ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase‐like 2 [L]) are efficacious in forming three‐dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5‐fold and 3‐fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5‐fold and 3‐fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.     

Platelet‐rich plasma enhances the integration of bioengineered cartilage with native tissue in an in vitro model

Current therapies for cartilage repair can be limited by an inability of the repair tissue to integrate with host tissue. Thus, there is interest in developing approaches to enhance integration. We have previously shown that platelet‐rich plasma (PRP) improves cartilage tissue formation. This raised the question as to whether PRP could promote cartilage integration . Chondrocytes were isolated from cartilage harvested from bovine joints, seeded on a porous bone substitute and grown in vitro to form an osteochondral‐like implant. After 7 days, the biphasic construct was soaked in PRP for 30 min before implantation into the core of a donut‐shaped biphasic explant of native cartilage and bone. Controls were not soaked in PRP. The implant–explant construct was cultured for 2–4 weeks. PRP‐soaked bioengineered implants integrated with host tissue in 73% of samples, whereas controls only integrated in 19% of samples. The integration strength, as determined by a push‐out test, was significantly increased in the PRP‐soaked implant group (219 ± 35.4 kPa) compared with controls (72.0 ± 28.5 kPa). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP‐treated implant group, compared with untreated controls. Immunohistochemical studies revealed that the integration zone contained collagen type II and aggrecan. The cells at the zone of integration in the PRP‐soaked group had a 3.5‐fold increase in matrix metalloproteinase‐13 gene expression compared with controls. These results suggest that PRP‐soaked bioengineered cartilage implants may be a better approach for cartilage repair due to enhanced integration.     

Jellyfish collagen matrices conserve the chondrogenic phenotype in two‐ and three‐dimensional collagen matrices

Cartilage is a tissue with a very low capability of self‐repair and the search for suitable materials supporting the chondrogenic phenotype and thus avoiding fibrotic dedifferentiation for matrix‐associated chondrocyte transplantation (MACI) is ongoing. Jellyfish collagen was thought to be a suitable material mainly because of its good availability and easy handling. Collagen was extracted from jellyfish Rhopilema esculentum and the spreading of porcine chondrocytes on two (2D) and three dimensional (3D) collagen matrices examined in comparison with vertebrate collagens, placenta collagen and a commercially available matrix from porcine collagen type I (Optimaix®). In 2D, most chondrocytes kept their round shape on jellyfish collagen and vertebrate collagen type II compared with vertebrate collagen type I. This was also confirmed in 3D experiments, where chondrocytes preserved their phenotype on jellyfish collagen, as indicated by high collagen II/(II + I) ratios (≥54 % and ~92 % collagen type II in mRNA and protein, respectively) and no proliferation during 28 days of cultivation. These observations were discussed with a view to potential structural differences of jellyfish collagen, which might influence the integrin‐mediated adhesion mechanisms of vertebrate cells on jellyfish collagen. This probably results from a lack of integrin‐binding sites and the existence of an alternative binding mechanism such that cells kept their round shape on jellyfish collagen, preventing chondrocytes from dedifferentiation. Thus, collagen from R. esculentum is a very suitable and promising material for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of chondrocyte sheets prepared using a co‐culture method with temperature‐responsive culture inserts

Conventional culture methods using temperature‐responsive culture dishes require 4–5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co‐culture) on temperature‐responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real‐time polymerase chain reaction and immunostaining of the three‐layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan‐1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A‐disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin‐1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co‐cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd.     

Enhanced treatment of articular cartilage defect of the knee by intra‐articular injection of Bcl‐xL‐engineered mesenchymal stem cells in rabbit model

Direct intra‐articular injection of mesenchymal stem cells (MSCs) has been proposed as a potential cell therapy for cartilage defects. This cell therapy relies on the survival of the implanted MSCs. However, the arduous local environment may limit cell viability after implantation, which would restrict the cells' regenerative capacity. Thus, it is necessary to reinforce the implanted cells against the unfavourable microenvironment in order to improve the efficacy of cell therapy. We examined whether the transduction of an anti‐apoptotic protein, Bcl‐xL, into MSCs could prevent cell death and improve the implantation efficiency of MSCs in a rabbit model. Our current findings demonstrate that the group treated with Bcl‐xL‐engineered MSCs could improve cartilage healing both morphologically and histologically when compared with the controls. These results suggest that intra‐articular injection of Bcl‐xL‐engineered MSCs is a potential non‐invasive therapeutic method for effectively treating cartilage defects of the knee. Copyright © 2009 John Wiley & Sons, Ltd.     

A nanofibrous cell‐seeded hydrogel promotes integration in a cartilage gap model

The presence of a defect in mature articular cartilage can lead to degenerative changes of the joint. This is in part caused by the inability of cartilage to regenerate tissue that is capable of spanning a fissure or crack. In this study, we hypothesized that introduction of a biodegradable cell‐seeded nanofibrous hydrogel, Puramatrix^?, into a cartilage gap would facilitate the generation of a mechanically stable interface. The effects of chondrocyte incorporation within the hydrogel and supplementation with transforming growth factor‐β3 (TGFβ3), a known regulator of cell growth and differentiation, on cartilage integration were examined mechanically and histologically as a function of cell density and incubation time. When supplemented with TGFβ3, the cell‐seeded hydrogel exhibited abundant matrix generation within the hydrogel and a corresponding increase in maximum push‐out stress as compared to all other groups. Furthermore, initial cell seeding density affected interfacial strength in a time‐dependent manner. This study suggests that a cell‐seeded TGFβ3‐supplemented hydrogel can encourage integration between two opposing pieces of articular cartilage. Copyright © 2009 John Wiley & Sons, Ltd.     

自体骨髓间充质干细胞修复兔关节软骨缺损的实验研究

目的通过对受损关节软骨进行未经诱导的兔自体骨髓间质干细胞移植,探讨组织工程移植修复兔关节软骨缺损的效果。方法取青紫蓝兔股骨骨髓分离间充质干细胞进行培养,采用变性处理及透明质酸盐修饰后的吸收性明胶海绵为载体,实验组用骨髓间充质干细胞修复自体股骨髁关节软骨缺损,对照组进行单纯吸收性明胶海绵移植。结果移植24周后,肉眼下实验组移植物与正常软骨组织难以区分,软骨表面光滑,对照组移植物为白色的疏松组织。光镜下修复组织类似周围正常软骨组织结构,甲苯胺蓝异染与正常软骨组织无区别,为透明软骨组织。对照组24周关节软骨缺损区被纤维软骨样组织修复。结论自体骨髓间质干细胞移植于受损关节软骨处,可促进关节缺损的修复,恢复软骨组织的结构功能。     

Mechanical stimulations on human bone marrow mesenchymal stem cells enhance cells differentiation in a three‐dimensional layered scaffold

Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane–strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.     

Regenerative effect of adipose tissue‐derived stem cells transplantation using nerve conduit therapy on sciatic nerve injury in rats

This study proposed a biodegradable GGT nerve conduit containing genipin crosslinked gelatin annexed with tricalcium phosphate (TCP) ceramic particles for the regeneration of peripheral nerves. Cytotoxicity tests revealed that GGT‐extracts were non‐toxic and promoted proliferation and neuronal differentiation in the induction of stem cells (i‐ASCs) derived from adipose tissue. Furthermore, the study confirmed the effectiveness of a GGT/i‐ASCs nerve conduit as a guidance channel in the repair of a 10‐mm gap in the sciatic nerve of rats. At eight weeks post‐implantation, walking track analysis showed a significantly higher sciatic function index (SFI) (P < 0.05) in the GGT/i‐ASC group than in the autograft group. Furthermore, the mean recovery index of compound muscle action potential (CMAP) differed significantly between GGT/i‐ASCs and autograft groups (P < 0.05), both of which were significantly superior to the GGT group (P < 0.05). No severe inflammatory reaction in the peripheral nerve tissue at the site of implantation was observed in either group. Histological observation and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/i‐ASCs nerve conduits were similar to those of the autografts. These promising results achieved through a combination of regenerative cells and GGT nerve conduits suggest the potential value in the future development of clinical applications for the treatment of peripheral nerve injury. Copyright © 2012 John Wiley & Sons, Ltd.     

Transplantation of cancerous cell sheets effectively generates tumour‐bearing model mice

Tumour‐bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene‐transfected mammary gland adenocarcinoma cells, 4T1‐luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature‐responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin‐based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase‐gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1‐luc2 engraftment efficiency in living mouse tissues at the initial stage by 13‐fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet‐transplanted mice also accelerated, and the mean tumour volume became 1116 mm³, which was 10 times larger than that in cell suspension‐transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet‐based transplantation is a promising method for efficiently creating cancer‐bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of layered chondrocyte sheets created in a co‐culture system with synoviocytes in a hypoxic environment

Endeavouring to repair and regenerate articular cartilage using cell sheets, we have previously established a co‐culture system of chondrocytes and synoviocytes, and have reported the successful and rapid production of chondrocyte sheets. In the present study, to examine the effects of oxygen concentration on the chondrocyte sheets, we co‐cultured human articular chondrocytes and human synoviocytes in 2%, 5% and 21% oxygen, and measured chondrocyte metabolic activity and proliferation activities under each condition for 14 days in culture. Layered chondrocyte sheets were also created under each condition and the proteoglycan (PG) level was compared with the gene expression of type I collagen (COL1), COL2, COL27, tissue metallopeptidase inhibitor 1 (TIMP1), fibronectin‐1 (FN1), SRY‐related HMG Box 9 (SOX9), aggrecan‐1 (ACAN), integrin‐α10 (ITGα10), matrix metalloproteinase 3 (MMP3), MMP13 and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5). Compared with 5% and 21% oxygen, the 2% condition caused significantly greater cell metabolic activity and proliferation (p < 0.05). The 2% condition produced a 10% greater PG level compared with 21% oxygen (p < 0.05). All conditions increased the expression of chondrocyte‐specific genes, such as COL2, and were associated with low expression levels of catabolic factors, such as MMP3 and MMP13. These observations indicated that the specificity of the chondrocyte sheets was maintained under all conditions. The culture times did not differ between the 5% and 21% conditions. Compared with 21% oxygen, layered chondrocyte sheets rich in extracellular matrix were created 2.85 days earlier in 2% oxygen, which is similar to the level found in deep cartilage. Copyright © 2016 John Wiley & Sons, Ltd.     

霉酚酸酯对小鼠异基因骨髓移植后急性移植物抗宿主病的预防作用

目的：探讨新型免疫抑制剂霉酚酸酯（MMF）对小鼠异基因骨髓移植（allo-BMT)后急性移植物抗宿主病（aGVHD)的预防作用。方法：用主要组织相容性抗原（MHC）完全不合的纯种近交系小鼠[供鼠：雌性C57BL／6J（H－2^b)鼠；受鼠：雄性BALB／c(H-2^d)鼠]建立allo-BMT/aGVHD动物模型，随机分6组，给予MMF、环孢菌素A（CsA) 甲氨喋呤（MTX）或联合不同剂量MMF作为aGVHD预防方案。观察其aGVHD的预防作用。结果：移植小鼠出现典型的aGVHD症状，未用aGVHD预防方案的移植小鼠（第6组）死亡高峰在移植后第5－第7天，死亡率达100％。用不同aGVHD预防方案的1－5组小鼠aGVHD症状明显减轻，平均生存时间（MST）较第6组显著延长（分别延长3．4，8．4，9．0，6．1，8．8d（P＜0．05）。不同aGVHD预防方案的移植小鼠之间，移植后外周血常规变化差异无显著性。经用各预防方案后，移植小鼠cGVHD病理表现减轻，且MMF、CsA、MTX三药联合组的病理分级轻于MMF单药组或CsA MTX标准方案组。结论：MMF单药及与CsA MTX联合均能有效预防小鼠allo-BMT后的aGVHD，减轻其症状和病理损害程度，显著延长平均生存时间。小剂量MMF（≤30mg/kg）与CsA TMX的联用效果优于大剂量MMF（60mg/kg）与CsA MTX的联用效果。适当剂量的MMF（10－60mg/kg）对骨髓植入和造血恢复没有显著影响。     

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.     

Lentiviral shRNA knock‐down of ADAMTS‐5 and ‐9 restores matrix deposition in 3D chondrocyte culture

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS‐1, ‐4, ‐5, ‐8, ‐9 and ‐15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell‐lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock‐down of ADAMTS‐5 and ‐9 expression in these cells and, by culturing them on three‐dimensional (3D) scaffolds, show that reduction in expression of ADAMTS‐5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue‐engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS‐5 may be a therapeutic target in the treatment of arthritic disease. Copyright © 2010 John Wiley & Sons, Ltd.