Cartilage tissue formation using redifferentiated passaged chondrocytes in vitro

Articular cartilage has limited ability for repair when damaged by trauma or degenerative disease, such as osteoarthritis, which can result in pain and compromised quality of life. Biological surface replacements developed using tissue engineering methods are a promising approach for cartilage repair, which would avoid the need for total joint replacement with the synthetic implants used currently. A basic requirement of in vitro tissue generation is a supply of sufficient number of cells, which are difficult to acquire from sparsely cellular cartilage tissue. Previously, we have shown that coculture of in vitro-expanded dedifferentiated chondrocytes (P2) with small numbers of primary chondrocytes (P0) induces redifferentiation in passaged (P2) cells. In this study we show that this redifferentiation is not a transient change. After 4 weeks of coculture, the P0 and P2 cells were separated by flow-associated cell sorting, and the redifferentiated P2 (dP2) were cultured alone for a further 4 weeks. The redifferentiated dP2 cells formed thicker cartilage tissue compared to the tissue generated by P2 cells. The newly formed tissue contained type II collagen as demonstrated by immunohistochemical staining and accumulated more proteoglycan per cell than the tissue formed by P2 cells. The dP2 cells also exhibited higher type II collagen and lower type I collagen gene expression than the P2 cells. Interestingly, dP2 cells were able to exert the same effect as P0 cells when cocultured with P2 cells. In conclusion, under proper culture conditions, redifferentiated passaged chondrocytes behave similarly to primary chondrocytes. This coculture system approach can be used to increase the number of differentiated chondrocytes that can be obtained by classical monolayer cell expansion and represents a novel way to acquire sufficient cell numbers for cartilage tissue engineering.     

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.     

Retaining zonal chondrocyte phenotype by means of novel growth environments

The loss of phenotype in articular chondrocytes expanded in monolayer has been established as a possible contributor to the deficiencies associated with in vitro cartilage engineering and autologous cell transplantation procedures. We cultured zonal articular chondrocytes on tissue culture plastic, collagen II-coated polystyrene, and aggrecan-coated polystyrene in an effort to find a surface that can either prevent or slow the loss of phenotype. In addition, we encapsulated passaged cells in agarose to examine the effect of three-dimensional culture on redifferentiating zonal chondrocytes. We used real-time polymerase chain reaction to measure the relative gene expression levels of collagen I and II, aggrecan, and superficial zone protein over relevant passages (P0-P4). Results showed that tissue culture plastic and the collagen II-coated surface induced rapid loss of phenotype in zonal articular chondrocytes. The aggrecan-coated surface had a less detrimental effect on the chondrocytic phenotype of seeded cells, inducing gene expression characteristics comparable to those of agarose-encapsulated cells. Furthermore, when chondrocytes that had been previously passaged on a collagen II surface were placed on an aggrecan surface, the zonal cells showed a dramatic change in gene expression from fibroblastic to chondrocytic. These results indicate that a culture environment using aggrecan as a substratum or agarose as a scaffold is crucial to the development of phenotypically correct articular cartilage.     

Passaged Goat Costal Chondrocytes Provide a Feasible Cell Source for Temporomandibular Joint Tissue Engineering

Costal cartilage is commonly harvested for various types of facial reconstructive surgery. The ability of costal chondrocytes (CCs) to produce relevant extracellular matrix, including glycosaminoglycans (GAGs) and collagens, makes them an appealing cell source for fibrocartilage engineering. In order to obtain enough cells for tissue engineering, however, cell expansion will likely be necessary. This study examined CCs at passages 0, 1, 3, and 5, as well as temporomandibular (TMJ) disc cells, in a scaffoldless tissue engineering approach. It was hypothesized that earlier passage constructs would have more cartilaginous proteins and less fibrocartilaginous proteins. TMJ disc constructs had over twice the collagen content of any other group, as well as the largest tensile properties; however, the substantial contraction of the constructs and limited cell numbers make it a non-feasible cell source for tissue engineering. In general, statistical differences in mechanical properties or collagen content of the various CC groups were not observed; however, significantly more GAG was produced in the passaged CCs than the primary CCs. More collagen type II was also observed in some of the passaged groups. These results suggest not only feasibility but potential superiority of passaged CCs over primary CCs, which may lead to functional engineered fibrocartilage.     

Ⅱ型胶原酶消化法可短时间获得大量纯化大鼠关节软骨细胞

背景：建立一种经济、快捷、切实可行的软骨细胞分离培养体系对于软骨体外实验研究有着重要的意义。 目的：探讨与改进大鼠关节软骨细胞的培养方法。 方法：无菌条件下取新生1周龄SD雄性大鼠双侧髋及膝关节软骨,采用Ⅱ型胶原酶消化法分离软骨细胞并进行原代、传代培养及鉴定。 结果与结论：倒置相差显微镜下见原代培养的软骨细胞12 h后开始贴壁,3 d左右可形成单层,4 d左右即可传代。传至第6代后,部分细胞变为梭形;第7代后,绝大部分细胞变为长梭形和不规则形状,增殖能力减弱。甲苯胺蓝染色显示培养的软骨细胞核呈异染性,免疫荧光染色显示培养的软骨细胞Ⅱ型胶原呈阳性表达。说明采用此方法可在短时间内获得大量纯化的大鼠软骨细胞。     

以交联透明质酸钠为支架体外构建组织工程软骨

背景：采用组织工程技术再生和重建软骨是目前修复软骨组织缺损效果最好、最有应用前景的方法。 目的：以体外培养的软骨细胞和交联透明质酸钠为支架材料,开发一套体外构建组织工程软骨的完整方案。 方法：分离新西兰兔膝关节软骨细胞,制成细胞悬液滴加于交联透明质酸钠支架上,体外复合培养21 d,提取RNA进行RT-PCR检测,制备冰冻切片进行显微观察和免疫组织化学观察。 结果与结论：软骨细胞接种于交联透明质酸钠支架材料后,可贴附于支架上生长,并且大量细胞聚集成团,在支架材料的纤维间隙中生长或呈单层细胞附着于支架材料纤维。细胞-支架复合物表达软骨组织特异性蛋白聚糖基因和Ⅱ型胶原α1基因,以及软骨组织特异性蛋白Ⅱ型胶原蛋白,可维持软骨细胞表型。表明培养的细胞-支架复合物在体外培养可形成软骨细胞外基质,有望获得组织工程软骨组织。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

Gene expression profiling of human articular cartilage grafts generated by tissue engineering

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. In this study autologous human cartilage tissue engineering grafts based on bioresorbable polyglactin/polydioxanone scaffolds were analyzed on the broad molecular level. RNA from freshly isolated, primary and expanded adult articular chondrocytes and from three-dimensional cartilage grafts were used for gene expression profiling using oligonucleotide microarrays. The capacity of cartilage grafts to form cartilage matrix was evaluated after subcutaneous transplantation into nude mice. Gene expression profiling showed reproducibly the regulation of 905 genes and documented that chondrocytes undergo fundamental changes during cartilage tissue engineering regarding chondrocyte metabolism, growth, and differentiation. Three-dimensional assembly of expanded, dedifferentiated chondrocytes initiated the re-differentiation of cells that was accompanied by the reversal of the expression profile of multiple players of the transforming growth factor (TGF) signaling pathway including growth and differentiation factor-5 and inhibitor of differentiation-1 as well as by the induction of typical cartilage-related matrix genes such as type II collagen and cartilage oligomeric matrix protein. Cartilage grafts formed a cartilaginous matrix after transplantation into nude mice. Three-dimensional tissue culture of expanded articular chondrocytes initiates chondrocyte re-differentiation in vitro and leads to the maturation of cartilage grafts towards hyaline cartilage in vivo.     

Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture

Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non‐chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation‐associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast‐like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.     

Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels

Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications.     

Chondrocyte redifferentiation in 3D: the effect of adhesion site density and substrate elasticity

To obtain sufficient cell numbers for cartilage tissue engineering with autologous chondrocytes, cells are typically expanded in monolayer culture. As a result, they lose their chondrogenic phenotype in a process called dedifferentiation, which can be reversed upon transfer into a 3D environment. We hypothesize that the properties of this 3D environment, namely adhesion site density and substrate elasticity, would influence this redifferentiation process. To test this hypothesis, chondrocytes were expanded in monolayer and their phenotypical transition was monitored. Agarose hydrogels manipulated to give different RGD adhesion site densities and mechanical properties were produced, cells were incorporated into the gels to induce redifferentiation, and constructs were analyzed to determine cell number and extracellular matrix production after 2 weeks of 3D culture. The availability of adhesion sites within the gels inhibited cellular redifferentiation. Glycosaminoglycan production per cell was diminished by RGD in a dose-dependent manner and cells incorporated into gels with the highest RGD density, remained positive for collagen type I and produced the least collagen type II. Substrate stiffness, in contrast, did not influence cellular redifferentiation, but softer gels contained higher cell numbers and ECM amounts after 2 weeks of culture. Our results indicate that adhesion site density but not stiffness influences the redifferentiation process of chondrocytes in 3D. This knowledge might be used to optimize the redifferentiation process of chondrocytes and thus the formation of cartilage-like tissue.     

Comparative phenotypic analysis of articular chondrocytes cultured within type I or type II collagen scaffolds

Among the existing repair strategies for cartilage injury, tissue engineering approach using biomaterials and chondrocytes offers hope for treatments. In this context, collagen-based biomaterials are good candidates as scaffolds for chondrocytes in cell transplantation procedures. These scaffolds are provided under different forms (gel or crosslinked sponge) made with either type I collagen or type I or type II atelocollagen molecules. The present study was undertaken to investigate how bovine articular chondrocytes sense and respond to differences in the structure and organization of these collagen scaffolds, over a 12-day culture period. When chondrocytes were seeded in the collagen scaffolds maintained in free-floating conditions, cells contracted gels to 40-60% and sponges to 15% of their original diameter. Real-time polymerase chain reaction analysis indicated that the chondrocyte phenotype, assessed notably by the ratio of COL2A1/COL1A2 mRNA and alpha10/alpha11 integrin subunit mRNA, was comparatively better sustained in type I collagen sponges when seeded at high cell density, also in type I atelocollagen gels. Besides, proteoglycan accumulation in the different scaffolds, as assessed by measuring the sulfated glycosaminoglycan content, was found be highest in type I collagen sponges seeded at high cell density. In addition, gene expression of matrix metalloproteinase-13 increased dramatically (up to 90-fold) in chondrocytes cultured in the different gels, whereas it remained stable in the sponges. Our data taken together reveal that type I collagen sponges seeded at high cell density represent a suitable material for tissue engineering of cartilage.     

Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression

To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous type II collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the type II collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous type II collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.     

Elastic cartilage engineering using novel scaffold architectures in combination with a biomimetic cell carrier

Tissue engineering of an elastic cartilage graft that meets the criterion for both structural and functional integration into host tissue, as well as allowing for a clinically tolerable immune response, is a challenging endeavour. Conventional scaffold technologies have limitations in their ability to design and fabricate complex-shaped matrix architectures of structural and mechanical equivalence to elastic cartilage found in the body. We attempted to investigate the potential of conventionally isolated and passaged chondrocytes (2D environment) when seeded and cultured in combination with a biomimetic hydrogel in a mechanically stable and biomimetic composite matrix to form elastic cartilage within ectopic implantation sites. In vitro cultured scaffold/hydrogel/chondrocytes constructs showed islets of cartilage and mineralized tissue formation within the cell-seeded specimens in both pig and rabbit models. Specimens with no cells seeded showed only vascularized fibrous tissue ingrowth. These studies demonstrated the potential of such scaffold/hydrogel/cell constructs to support chondrogenesis in vivo. However, it also showed that even mechanically stable scaffolds do not allow regeneration of a large mass of structural and functional cartilage within a matrix architecture seeded with 2D passaged chondrocytes in combination with a cell biomimetic carrier. Hence, future experiments will be designed to evaluate an initial 3D culture of chondrocytes, effect on cell phenotype and their subsequent culture within biomimetic 3D scaffold/cell constructs.     

永生化下颌骨髁突软骨细胞的微囊化研究

为了探讨微囊包裹软骨细胞在软骨组织工程中的适用性 ,根据气流切割原理采用海藻酸钠 -多聚赖氨酸 -海藻酸钠 ( APA)对永生化下颌骨髁突软骨细胞 ( Im mortalized mandibular condylar chondrocyte,IMCC)进行微囊包裹。用倒置显微镜观察、台盼蓝染色、细胞记数、HE染色、免疫组化等方法检测微囊的大小、细胞的生长及微囊内组织的软骨特性等情况。研究发现 ,IMCC可在微囊内存活 ,活细胞率 >80 % ,微囊直径平均 779μm。细胞数量随着培养时间的延长逐渐增多 ,约 2 0 d左右达到平台期 ,细胞在囊内呈簇样生长 ,高表达软骨特异的蛋白多糖和 型胶原。提示 IMCC可在微囊内形成类软骨组织样结构 ,微囊技术适用于包裹软骨细胞     

Designing zonal organization into tissue-engineered cartilage

Cartilage tissue engineering strategies generally result in homogeneous tissue structures with little resemblance to the native zonal organization of articular cartilage. The objective of this study was to use bilayered photopolymerized hydrogels to organize zone-specific chondrocytes in a stratified framework and study the effects of this three-dimensional coculture system on the properties of the engineered tissue. Superficial and deep zone chondrocytes from bovine articular cartilage were photoencapsulated in separate hydrogels as well as in adjacent layers of a bilayered hydrogel. Histology, mechanical testing, and biochemical analysis was performed after culturing in vitro. To evaluate the influence of coculture on tissue properties, the layers were separated and compared to constructs containing only superficial or deep cells. In the bilayered constructs, deep cells produced more collagen and proteoglycan than superficial cells, resulting in cartilage tissue with stratified, heterogeneous properties. Deep cells cocultured with superficial cells in the bilayered system demonstrated reduced proliferation and increased matrix synthesis compared to deep cells cultured alone. The bilayered constructs demonstrated greater shear and compressive strength than homogenous cell constructs. This study demonstrated that interactions between zone-specific chondrocytes affect the biological and mechanical properties of engineered cartilage. Strategies aimed to structurally organize zone-specific cells and encourage heterotypic cell interactions may contribute to improved functional properties of engineered cartilage.     

Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity

For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.     

Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.     

Subculture of chondrocytes on a collagen type I-coated substrate with suppressed cellular dedifferentiation

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.