Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage‐specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three‐dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D‐cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage‐specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region. Copyright © 2012 John Wiley & Sons, Ltd.     

Efficacy of thermoresponsive,photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin‐soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell‐mediated contraction, as compared against non‐methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell–matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non‐methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non‐methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non‐methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin‐solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal‐ and light‐induced polymerization but compromises chondrogenic differentiation of seeded MSCs.     

Conditioned medium as a strategy for human stem cells chondrogenic differentiation

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow‐derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly‐derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three‐dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage‐related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Macromolecular crowding as a means to assess the effectiveness of chondrogenic media

Chondrocyte‐based tissue engineering requires in vitro cell expansion, which is associated with phenotypic losses, decrease in Collagen Type II synthesis and increase in Collagen Type I synthesis. Another major obstacle in clinical translation of chondrocyte‐based therapies is the lack of extracellular matrix (ECM) in the engineered cartilage substitutes. Various research and commercially available media claim that they can maintain chondrogenic phenotype, whereas macromolecular crowding (MMC) has been shown to increase tissue‐specific ECM deposition and maintain cell phenotype in vitro. Herein, we hypothesised that the combination of chondrogenic media with MMC will enable chondrogenic phenotype maintenance during in vitro expansion and increase cartilage‐specific ECM deposition, enabling that way the development of a tissue‐engineered cartilage substitute. Immunocytochemistry analysis of Passage 3 human chondrocytes in normal media in monolayer revealed that MMC significantly increased Collagen Type I deposition, whereas no statistical difference was observed in Collagen Type II deposition. When Passage 3 human chondrocytes were cultured in normal media and alginate beads, immunocytochemistry analysis revealed that MMC increased, albeit not significantly, both Collagen Type I and Collagen Type II deposition. Subsequently, human chondrocytes were expanded up to Passage 6 in either fetal bovine serum or human serum and redifferentiated using commercially available chondrogenic media in either monolayer or alginate beads. Immunocytochemistry analysis revealed that MMC, independently of the serum used, significantly increased Collagen Type I deposition in human‐redifferentiated monolayer and alginate bead chondrocyte cultures, whereas almost no Collagen Type II was detected. These data clearly illustrate that an optimal chondrogenic medium is still elusive.     

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.     

Comprehensive characterization of chondrocyte cultures in plasma and whole blood biomatrices for cartilage tissue engineering

Many synthetic polymers and biomaterials have been used as matrices for 3D chondrocyte seeding and transplantation in the field of cartilage tissue engineering. To develop a fully autologous carrier for chondrocyte cultivation, we examined the feasibility of allogeneic plasma and whole blood‐based matrices and compared them to agarose constructs. Primary articular chondrocytes isolated from 12‐month‐old pigs were embedded into agarose, plasma and whole blood matrices and cultivated under static‐free swelling conditions for up to four weeks. To evaluate the quality of the synthesized extracellular matrix (ECM), constructs were subjected to weekly examinations using histological staining, spectrophotometry, immunohistochemistry and biochemical analysis. In addition, gene expression of cartilage‐specific markers such as aggrecan, Sox9 and collagen types I, II and X was determined by RT‐PCR. Chondrocyte morphology was assessed via scanning electron microscopy and viability staining, including proliferation and apoptosis assays. Finally, ¹³ C NMR spectroscopy provided further evidence of synthesis of ECM components. It was shown that chondrocyte cultivation in allogeneic plasma and whole‐blood matrices promoted sufficient chondrocyte viability and differentiation behaviour, resulting in neo‐formation of a hyaline‐like cartilage matrix. Copyright © 2012 John Wiley & Sons, Ltd.     

Adseverin,an actin binding protein,regulates articular chondrocyte phenotype

Chondrocytes dedifferentiate as a result of monolayer culture for cell number expansion. This is associated with the development of an elongated shape, increased actin polymerization, development of stress fibres, and expression of contractile molecules. Given the changes in actin status with dedifferentiation, the hypothesis of this study was that adseverin, an actin severing and capping protein, plays a role in regulating chondrocyte phenotype and function. This study reports that serial passaging of articular chondrocytes in monolayer culture resulted in loss of adseverin protein expression as early as Day 14 of culture and remained repressed in Passage 2 (P2) cells. Knockdown of adseverin by siRNA in primary chondrocytes promoted an increase in cell size and an elongated shape, actin stress fibres, decreased G‐/F‐actin ratio, and increased number of actin‐free barbed ends. The cells also showed increased expression of the contractile genes and proteins, vinculin and α‐smooth muscle actin, and increased ability to contract collagen gels. These are all features of dedifferentiation. These effects were due to adseverin as adseverin overexpression following transfection of the green fluorescent protein‐adseverin plasmid partially reversed all of these changes in P2 chondrocytes. Furthermore, sox9 and aggrecan chondrogenic gene expression was upregulated, and collagen type I genes expression was downregulated with adseverin overexpression. The change in aggrecan mRNA expression had functional consequence as these cells exhibited increased total proteoglycan synthesis. These findings demonstrate that adseverin regulates features indicative of redifferentiation in passaged articular chondrocytes through modulation of the actin cytoskeleton status and potentially may regulate the maintenance of phenotype in primary chondrocytes.     

Human articular chondrocytes on macroporous gelatin microcarriers form structurally stable constructs with blood‐derived biological glues in vitro

Biodegradable macroporous gelatin microcarriers fixed with blood‐derived biodegradable glue are proposed as a delivery system for human autologous chondrocytes. Cell‐seeded microcarriers were embedded in four biological glues—recalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue—and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood‐derived alternatives. Five further experiments were conducted with recalcified citrated platelet‐rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers supported chondrocyte adhesion and expansion as well as extracellular matrix (ECM) synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of ECM proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed the presence of SOX‐9‐ and S‐100‐positive chondrocytes as well as the production of aggrecan and collagen type I, but not of the cartilage‐specific collagen type II. These results imply that blood‐derived glues are indeed potentially applicable for encapsulation of chondrocyte‐seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Cell yield,chondrogenic potential,and regenerated cartilage type of chondrocytes derived from ear,nasoseptal, and costal cartilage

Functional reconstruction of large cartilage defects in subcutaneous sites remains clinically challenging because of limited donor cartilage. Tissue engineering is a promising and widely accepted strategy for cartilage regeneration. To date, however, this strategy has not achieved a significant breakthrough in clinical translation owing to a lack of detailed preclinical data on cell yield and functionality of clinically applicable chondrocytes. To address this issue, the current study investigated the initial cell yield, proliferative potential, chondrogenic capacity, and regenerated cartilage type of human chondrocytes derived from auricular, nasoseptal, and costal cartilage using a scaffold‐free cartilage regeneration model (cartilage sheet). Chondrocytes from all sources exhibited high sensitivity to basic fibroblast growth factor within 8 passages. Nasoseptal chondrocytes presented the strongest proliferation rate, whereas auricular chondrocytes obtained the highest total cell amount using comparable cartilage sample weights. Importantly, all chondrocytes at fifth passage showed strong chondrogenic capacity both in vitro and in the subcutaneous environment of nude mice. Although some significant differences in histological structure, cartilage matrix content and cartilage type specific proteins were observed between the in vitro engineered cartilage and original tissue; the in vivo regenerated cartilage showed mature cartilage features with high similarity to their original native tissue, except for minor matrix changes influenced by the in vivo environment. The current study provides detailed preclinical data for choice of chondrocyte source and thus promotes the clinical translation of cartilage regeneration approach.     

Effects of mechanical loading on collagen propeptides processing in cartilage repair

Injured articular cartilage has poor reparative capabilities and if left untreated may develop into osteoarthritis. Unsatisfactory results with conventional treatment methods have brought as an alternative treatment the development of matrix autologous chondrocyte transplants (MACTs). Recent evidence proposes that the maintenance of the original phenotype by isolated chondrocytes grown in a scaffold transplant is linked to mechanical compression, because macromolecules, particularly collagen, of the extracellular matrix have the ability to ‘self‐assemble’. In load‐bearing tissues, collagen is abundantly present and mechanical properties depend on the collagen fibre architecture. Study of the active changes in collagen architecture is the focus of diverse fields of research, including developmental biology, biomechanics and tissue engineering. In this review, the structural model of collagen assembly is presented in order to understand how scaffold geometry plays a critical role in collagen propeptide processing and chondrocyte development. When physical forces are applied to different cell‐based scaffolds, the resulting specific twist of the scaffolds might be accompanied by changes in the fibril pattern synthesis of the new collagen. The alteration in the scaffolds due to mechanical stress is associated with cellular signalling communication and the preservation of N‐terminus procollagen moieties, which would regulate both the collagen synthesis and the diameter of the fibre. The structural difference would also affect actin stabilization, cytoskeleton remodelling and proteoglycan assembly. These effects seemed to be dependent on the magnitude and duration of the physical stress. This review will contribute to the understanding of mechanisms for collagen assembly in both a natural and an artificial environment. Copyright © 2009 John Wiley & Sons, Ltd.     

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue‐engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real‐time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round‐shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real‐time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Scaffold‐free 3D cellulose acetate membrane‐based cultures form large cartilaginous constructs

Scaffold‐free three‐dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold‐free 3D membrane‐based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane‐based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin‐O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α₁ (COL2A1). Membrane‐based cultures (P1) formed flexible disc‐like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin‐O and collagen type II staining was found in membrane‐based and pellet cultures at P1–3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane‐based cultures at P1. After 21 days the membrane‐based cultures did not express COL2A1. We concluded that membrane‐based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane‐based cultures provided the highest COL2A1 expression and a large, smooth and cartilage‐like surface. As these are appropriate features for clinical applications, we assume that membrane‐based cultures might be of use in cartilage regeneration if they displayed similar results in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Extracellular matrix enhances differentiation of adipose stem cells from infrapatellar fat pad toward chondrogenesis

The objective was to improve proliferation and chondrogenic potential of adipose stem cells (ASCs) by expansion on extracellular matrix (ECM) deposited by either ASCs or synovium‐derived stem cells (SDSCs). ASCs isolated from porcine infrapatellar fat pad were separately expanded on conventional plastic flasks, ASC‐deposited ECM and SDSC‐deposited ECM. ASCs were centrifuged to form pellets and cultured in a serum‐free chondrogenic medium with either TGFβ3 or TGFβ3 combined with BMP‐6. Cell number yielded on ECM expansion did not show a significant difference in deposition between ASCs and SDSCs but was 6–10 times that grown on non‐coated flasks. ECM‐expanded ASCs exhibited a lower level of intracellular reactive oxygen species (ROS) compared to those grown on non‐coated flasks. Typical chondrogenic markers, including type II collagen and glycosaminoglycans (GAGs), were intensively distributed in the pellets from ECM‐expanded ASCs instead of those from flask‐grown cells. ASCs expanded on ECM, either from ASCs or SDSCs, exhibited a similar chondrogenic index (GAG:DNA), which was significantly higher than that from ASCs grown on non‐coated flasks. The combination of TGFβ3 and BMP‐6 increased 36% more in ASC chondrogenic index than the treatment with TGFβ3 alone. Interestingly, ECM pretreatment also decreased expanded ASC hypertrophic marker genes. ECM deposited by either ASCs or SDSCs did not exhibit enhanced adipogenic differentiation of ASCs. Our study indicates that the sequential application of ECM for cell expansion and combined TGFβ3 with BMP‐6 for chondrogenic differentiation may be a promising approach for ASC‐based cartilage tissue engineering and regeneration. Copyright © 2011 John Wiley & Sons, Ltd.     

The fate of bovine bone marrow stromal cells in hydrogels: a comparison to nucleus pulposus cells and articular chondrocytes

Bone marrow‐derived stromal cells (BMSCs) are good candidates for cell‐based tissue regeneration. For such purposes, cell survival within three‐dimensional (3D) scaffolds is often desirable. We hypothesize that undifferentiated BMSCs will have difficulties thriving within these gels, in contrast to articular chondrocytes (ACs) and nucleus pulposus cells (NPCs), but that early chondrogenic differentiation of the former will increase their survival. BMSCs, ACs and NPCs cast in 1.2% alginate or 2% agarose were cultured for 21 days in serum‐containing media. BMSCs were also cultured in medium with 10 ng/ml TGF‐β1. By day 21, NPCs and ACs proliferated, maintained upregulation of aggrecan and collagen type II, produced glycosaminoglycans and stained positively for collagen type II in both scaffolds. In contrast, the number of living BMSCs and the DNA content of their constructs decreased in both scaffolds. Addition of TGF‐β₁ resulted in cell survival and behaviour more similar (gene expression, glycosaminoglycan production and collagen type II synthesis) to ACs and NPCs. This study demonstrated that, unlike ACs and NPCs, undifferentiated BMSCs have more difficulty thriving within hydrogels, but that this can be improved by chondrogenic induction. Hence, immediate conditioning of BMSCs could be a worthwhile strategy. Copyright © 2009 John Wiley & Sons, Ltd.     

兔关节软骨细胞在藻酸盐串珠中培养维持其表型稳定的研究

目的:软骨细胞在单层培养时,多次传代后细胞表型发生改变。将兔关节软骨细胞在藻酸盐串珠中作立体培养,以保持其特有表型。方法:用酶消化法获取兔关节软骨细胞,分别在普通培养瓶中作贴壁的单层培养,并传代;或制成细胞/藻酸盐悬液,再进一步制成串珠,使细胞在具有三维立体结构的串珠中生长、繁殖。细胞涂片、石蜡切片,爱尔新蓝染色,采用倒置显微镜、透射电镜观察;以RT-PCR方法检测软骨细胞中II型胶原及凝集聚糖mRNA的表达。结果:单层培养时有较高的细胞增殖率,5代以后渐失去软骨细胞特有的表型。立体培养时细胞分泌的基质大部分位于自身的周围,特有表型可长期保持稳定。3个月后藻酸盐串珠中的软骨细胞仍可测得II型胶原和凝集聚糖的表达。结论:软骨细胞在藻酸盐串珠中培养有助于其合成、分泌基质,维持细胞特有表型的稳定。     

The influence of matrix elasticity on chondrocyte behavior in 3D

Cells actively probe the stiffness of their surrounding and respond to it. The authors recently found that maintenance of the chondrogenic phenotype was directly influenced by this property in 2D. Since studies about this process in 3D are still largely absent, this study aimed to transfer this knowledge into a 3D environment. Agarose was modified with RGD to allow active stiffness sensing or RGE as a control. Hydrogels with different mechanical properties were produced by using different concentrations of agarose. Primary chondrocytes were incorporated into the gel, cultured for up to two weeks, and then constructs were analyzed. Cells were surrounded by their own ECM from an early stage and maintained their chondrogenic phenotype, independent of substrate composition, as indicated by a high collagen type II and a lack of collagen type I production. However, softer gels showed higher DNA and GAG content and larger cell clusters than stiff gels in both RGD‐ and RGE‐modified agarose. The authors hypothesize that matrix elasticity in the tested range does not influence the maintenance of the chondrogenic phenotype in 3D but rather the size of the formed cell ECM clusters. The deviation of these findings from previous results in 2D stresses the importance of moving towards 3D systems that more closely mimic in vivo conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

Contraction‐induced Mmp13 and −14 expression by goat articular chondrocytes in collagen type I but not type II gels

Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we investigated whether the presence of collagen type I or II in gel lattices affects matrix contraction and relative gene expression levels of matrix proteins, MMPs and the subsequent degradation of collagen by goat articular chondrocytes. Only floating collagen I gels, and not those attached or composed of type II collagen, contracted during a culture period of 12 days. This coincided with an upregulation of both Mmp13 and ?14 gene expression, whereas Mmp1 expression was not affected. The release of hydroxyproline in the culture medium, indicating matrix degradation, was increased five‐fold in contracted collagen I gels compared to collagen II gels without contraction. Furthermore, blocking contraction of collagen I gels by cytochalasin B inhibited Mmp13 and ?14 expression and the release of hydroxyproline. The expression of cartilage‐specific ECM genes was decreased in contracted collagen I gels, with increased numbers of cells with an elongated morphology, suggesting that matrix contraction induces dedifferentiation of chondrocytes into fibroblast‐like cells. We conclude that the collagen composition of the gels affects matrix contraction by articular chondrocytes and that matrix contraction induces an increased Mmp13 and ?14 expression as well as matrix degradation. Copyright © 2011 John Wiley & Sons, Ltd.