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.     

Biomechanical analysis of a chondrocyte-based repair model of articular cartilage.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.     

Phenotypic characterization of human chondrocyte cell line C-20/A4: a comparison between monolayer and alginate suspension culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.     

Karyotyping of human chondrocytes in scaffold-assisted cartilage tissue engineering

Scaffold-assisted autologous chondrocyte implantation (ACI) is an effective clinical procedure for cartilage repair. The aim of our study was to evaluate the chromosomal stability of human chondrocytes subjected to typical cell culture procedures needed for regenerative approaches in polymer-scaffold-assisted cartilage repair. Chondrocytes derived from post mortem donors and from donors scheduled for ACI were expanded, cryopreserved and re-arranged in polyglycolic acid (PGA)-fibrin scaffolds for tissue culture. Chondrocyte redifferentiation was analyzed by electron microscopy, histology and gene expression analysis. Karyotyping was performed using GTG banding and fluorescence in situ hybridization on a single cell basis. Chondrocytes showed de- and redifferentiation accompanied by the formation of extracellular matrix and induction of typical chondrocyte marker genes like type II collagen in PGA-fibrin scaffolds. Post mortem chondrocytes showed up to 1.7% structural and high numbers of numerical (up to 26.7%) chromosomal aberrations, while chondrocytes from living donors scheduled for ACI showed up to 1.8% structural and up to 1.3% numerical alterations. Cytogenetically, cell culture procedures and PGA-fibrin scaffolds did not significantly alter chromosomal integrity of the chondrocyte genome. Human chondrocytes derived from living donors subjected to regenerative medicine cell culture procedures like cell expansion, cryopreservation and culture in resorbable polymer-based scaffolds show normal chromosomal integrity and normal karyotypes.     

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.     

软骨细胞微载体（Cytodex-3）平板三维培养扩增的实验研究

目的通过在微载体上进行平板培养扩增软骨细胞,并结合液态壳聚糖构建组织工程软骨。方法比较兔软骨细胞在单层培养与微载体上进行三维培养扩增软骨细胞的再分化能力。通过酶解法消化幼兔膝关节软骨后,得到种子细胞,分别进行单层和微载体三维培养扩增。通过评价细胞活性,倍增时间分析培养效果。并进行体外球型培养评价软骨细胞再分化能力,进行了糖胺多糖的定量生化分析。三维培养扩增软骨细胞与壳聚糖复合构建组织工程软骨,培养21天后通过组织学特种染色鉴定构建组织特性。结果微载体培养的软骨细胞可以保持良好活力和再分化能力,与单层培养体系相比较,糖胺多糖的定量生化分析（30．417±1．116ugGAG／mg样本）和（45．122±1．239ugGAG／mg样本）的差异具有统计学意义（P〈0．05）。结论在微载体上进行三维培养扩增软骨细胞可以加强细胞再分化能力。软骨细胞与壳聚糖合成后,可以在体外形成形态稳定的组织工程软骨。     

In vivo cultivation of human articular chondrocytes in a nude mouse-based contained defect organ culture model

The nude mouse model is an established method to cultivate and investigate tissue engineered cartilage analogues under in vivo conditions. One limitation of this common approach is the lack of appropriate surrounding articular tissues. Thus the bonding capacity of cartilage repair tissue cannot be evaluated. Widely applied surgical techniques in cartilage repair such as conventional and three-dimensional autologous chondrocyte implantation (ACI) based on a collagen gel matrix cannot be included into nude mouse studies, since their application require a contained defect. The aim of this study is to apply an organ culture defect model for the in vivo cultivation of different cell-matrix-constructs.Cartilage defects were created on osteochondral specimens which had been harvested from 10 human knee joints during total knee replacement. Autologous chondrocytes were isolated from the cartilage samples and cultivated in monolayer until passage 2. On each osteochondral block defects were treated either by conventional ACI or a collagen gel seeded with autologous chondrocytes, including a defect left empty as a control. The samples were implanted into the subcutaneous pouches of nude mice and cultivated for six weeks. After retrieval, the specimens were examined histologically, immunohistochemically and by cell morphology quantification.In both, ACI and collagen gel based defect treatment, a repair tissue was formed, which filled the defect and bonded to the adjacent tissues. The repair tissue was immature with low production of collagen type II. In both groups redifferentiation of chondrocytes remained incomplete. Different appearances of interface zones between the repair tissue and the adjacent cartilage were found.The presented contained defect organ culture model offers the possibility to directly compare different types of clinically applied biologic cartilage repair techniques using human articular tissues in a nude mouse model.     

Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor

This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel. Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks. Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy. During serial monolayer cultures chondrocytes dedifferentiated to a "fibroblast-like" structure and produced predominantly collagen type I. When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time. Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected. Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils. This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.     

Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage

The aim of the present study was to identify and characterize progenitor properties of human articular chondrocytes selected by using agarose suspension culture. In this chondrogenic selective culture condition, about 3.6% of seeded surplus chondrocytes from patients undergoing articular chondrocyte transplantation proliferated and formed cell clusters after 6 weeks. Phase-contrast microscopy and transmission electron microscopy revealed four different types of cell clusters differing in cellular content and matrix production. Based on their morphological features, they were named the homogenous (H), the homogenous matrix (HM), the differentiated matrix (DM) and the differentiated (D) cell clusters. All cell clusters showed positive safranin O staining, and matrix was positive for antibodies detecting type II collagen and aggrecan. The clusters were further demonstrated to express the genes for fibroblast growth factor receptor 3, type IIA collagen and type IIB collagen, while type X collagen was not expressed. After subcloning, the H and HM clusters demonstrated the best proliferative capacity. Chondrocytes from these two cell clusters also showed phenotypic plasticity in chondrogenic, adipogenic as well as osteogenic assays. This study demonstrates that existing subpopulations of cells with chondroprogenitor properties can be isolated from human adult articular cartilage using agarose suspension cultures.     

Secretion of chondrocyte stimulating factor by macrophages as a result of activation with collagen and proteoglycans.

In this study, we have investigated the role of extracellular matrix components of connective tissue, collagen and proteoglycans, in the activation of rabbit macrophages and articular chondrocytes. Addition of type II collagen, in native or denatured form, or proteoglycan subunits, led to the activation of macrophages and release of chondrocyte stimulating factor. Type I collagen, when used at higher concentrations, was also capable of macrophage activation. The conditioned media of activated macrophages induced an increased protease synthesis by chondrocytes. Collagen or proteoglycan subunits did not have any direct stimulatory effect on chondrocytes. Intraperitoneal injections of collagen induced infiltration of macrophages into the peritoneal cavity. Although these macrophages failed to produce significant levels of chondrocyte stimulating factor by themselves, they could be activated in culture with lipopolysaccharides or collagen to produce chondrocyte stimulating factor. The chondrocytes, treated with the conditioned medium of activated macrophages (with lipopolysaccharides or cartilage matrix components), failed to survive in suspension cultures.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

In vitro cartilage tissue formation by Co-culture of primary and passaged chondrocytes

Passaging chondrocytes to increase cell number is one way to overcome the major limitation to cartilage tissue engineering, which is obtaining sufficient numbers of chondrocytes to form large amounts of tissue. Because neighboring cells can influence cell phenotype and because passaging induces dedifferentiation, we examined whether coculture of primary and passaged bovine articular chondrocytes in 3-dimensional culture would form cartilage tissue in vitro. Chondrocytes passaged in monolayer culture up to 4 times were mixed with primary (nonpassaged) chondrocytes (5-40% of total cell number) and grown on filter inserts for up to 4 weeks. Passaged cells alone did not form cartilage, but with the addition of increasing numbers of primary chondrocytes, up to 20%, there was an increase in cartilage tissue formation as determined histologically and biochemically and demonstrated by increasing proteoglycan and collagen accumulation. The passaged cells appeared to be undergoing redifferentiation, as indicated by up-regulation of aggrecan, type II collagen, and SOX9 gene expression and decreased type I collagen expression. This switch in collagen type was confirmed using Western blots. Confocal microscopy showed that fluorescently labeled primary cells were distributed throughout the tissue. This coculture approach could provide a new way to solve the problem of limited cell number for cartilage tissue engineering.     

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.     

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.     

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.     

Species variability in the differentiation potential of in vitro-expanded articular chondrocytes restricts predictive studies on cartilage repair using animal models

Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.     

Three-dimensional microenvironments retain chondrocyte phenotypes during proliferation culture

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.     

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.     

Expression and roles of connective tissue growth factor in Meckel's cartilage development.

Meckel's cartilage is a prominent feature of the developing mandible, but its formation and roles remain unclear. Because connective tissue growth factor (CTGF, CCN2) regulates formation of other cartilages, we asked whether it is expressed and what roles it may have in developing mouse Meckel's cartilage. Indeed, CTGF was strongly expressed in anterior, central, and posterior regions of embryonic day (E) 12 condensing Meckel's mesenchyme. Expression decreased in E15 newly differentiated chondrocytes but surged again in E18 hypertrophic chondrocytes located in anterior region and most-rostral half of central region. These cells were part of growth plate-like structures with zones of maturation resembling those in a developing long bone and expressed such characteristic genes as Indian hedgehog (Ihh), collagen X, MMP-9, and vascular endothelial growth factor. At each stage examined perichondrial tissues also expressed CTGF. To analyze CTGF roles, mesenchymal cells isolated from E10 first branchial arches were tested for interaction and responses to recombinant CTGF (rCTGF). The cells readily formed aggregates in suspension culture and interacted with substrate-bound rCTGF, but neither event occurred in the presence of CTGF neutralizing antibodies. In good agreement, rCTGF treatment of micromass cultures stimulated both expression of condensation-associated macromolecules (fibronectin and tenascin-C) and chondrocyte differentiation. Expression of these molecules and CTGF itself was markedly up-regulated by treatment with transforming growth factor-beta1, a chondrogenic factor. In conclusion, CTGF is expressed in highly dynamic manners in developing Meckel's cartilage where it may influence multiple events, including chondrogenic cell differentiation and chondrocyte maturation. CTGF may aid chondrogenesis by acting down-stream of transforming growth factor-beta and stimulating cell-cell interactions and expression of condensation-associated genes.