Fibroblast growth factor (FGF) 18 signals through FGF receptor 3 to promote chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.     

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.     

N-CAM is not required for initiation of secondary chondrogenesis: the role of N-CAM in skeletal condensation and differentiation.

Condensation precedes chondrogenic differentiation during development of primary cartilage. While neural cell adhesion molecule (N-CAM) enhances condensation, it is unclear whether N-CAM is also required for initiation of chondrogenic differentiation. In this study, the role of N-CAM in secondary chondrogenesis from periosteal cells of the quadratojugal (QJ) from embryonic chicks was studied using several in vitro approaches. The QJ is a membrane bone and so is not preceded by cartilage formation during development. However, QJ periosteal cells can differentiate into chondrocytes to form secondary cartilage in vivo. When QJ periosteal cells were enzymatically released and plated in low density monolayer, clonal or agarose cultures, chondrogenesis was initiated in the absence of N-CAM expression. Furthermore, overexpression of the N-CAM gene in periosteal cells in monolayer culture significantly reduced the number of chondrocyte colonies, suggesting that N-CAM inhibits secondary chondrogenesis. In contrast, and consistent with expression in vivo, N-CAM is expressed during osteogenesis from QJ periosteal cells and mandibular mesenchyme in vitro. These results are discussed in relation to the role of N-CAM in osteogenesis and in primary and secondary condensation.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

In VitroDifferentiation Potential of the Periosteal Cells from a Membrane Bone, the Quadratojugal of the Embryonic Chick

The quadratojugal (QJ) is a neural crest-derived membrane bone in the maxillary region of the avian head.In vivoits periosteum undergoes both osteogenesis to form membrane bone and chondrogenesis to form secondary cartilage. This bipotential property, which also exists in some other membrane bones, is poorly understood. The present study used cell culture to investigate the differentiation potential of QJ periosteal cells. Three cell populations were enzymatically released from QJ periostea and plated at different densities. Cell density greatly affected phenotypic expression and differentiation pathways. We found two culture conditions that favored osteogenesis and chondrogenesis, respectively. In micromass culture, the periosteal cells produced a layer of osteogenic cells that expressed alkaline phosphatase (APase) and secreted bony extracellular matrix (ECM). In contrast, low-density monolayer culture elicited chondrogenesis. Cells with pericellular refractile ECM and round shape appeared at 7 to 8 days and formed colonies later. The chondrogenic phenotype of these cells was confirmed by immunolocalization of type II collagen and Alcian blue staining of ECM. This result demonstrated that a fully expressed chondrogenic phenotype can be achieved from membrane bone periosteal cells in primary monolayer culture. Chondrogenesis requires a cell density lower than confluence and cannot be initiated in confluent cultures. Among the three cell populations, those cells from the outer layer have the highest growth rate and require the lowest initial plating density (below 5 × 10³cells/ml) to achieve chondrogenesis. Cells from the inner layer have the slowest growth rate and chondrify at the highest initial density (below 5 × 10⁴cells/ml). Chondrocytes from all populations express distinct phenotypic markers—APase and type I collagen—from initial chondrogenesis, but are not hypertrophic morphologically. Furthermore, the fact that chondrocytes arise within the same colony as APase-positive polygonal cells suggests that chondrocytes may differentiate from precursors related to the osteogenic cell lineage. This cell culture approach mimics secondary cartilage and membrane bone formationin vivo.     

Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.     

Chondrogenesis and developments in our understanding

Conditions affecting cartilage through damage or age-related degeneration pose significant challenges to individual patients and their healthcare systems. The disease burden will rise in the future as life expectancy increases. This has resulted in vigorous efforts to develop novel therapies to meet current and future needs. Due to the limited regenerative capacity of cartilage, in vitro tissue engineering techniques have emerged as the favoured technique by which to develop replacements. Tissue engineering is mainly concerned with developing cartilage replacements in the form of chondrocyte suspensions and three-dimensional scaffolds seeded with chondrocytes. One major limiting factor in the development of clinically useful cartilage constructs is our understanding of the process by which cartilage is formed, chondrogenesis. For example, techniques of culturing chondrocytes in vitro have been used for decades, resulting in chondrocyte-like cells which produce an extracellular matrix of similar composition to native cartilage, but with inferior physical properties. It has now been realised that one aspect of chondrogenesis which had been ignored was the physical context in which cartilage exists in vivo. This has resulted in the development of bioreactor systems which aim to introduce various physical stresses to engineered cartilage in a controlled environment. This has resulted in some improvements in the quality of tissue engineered cartilage. This is but one example of how the knowledge of chondrogenesis has been translated into research practice. This paper aims to review what is currently known about the process of chondrogenesis and discusses how this knowledge can be applied to tissue engineering.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/10T1/2, is pluripotent; exposure to azacytidine or to bone morphogenetic protein-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differentiation specifically to the cartilage lineage and at high yields. These conditions include high cell density micromass cultures, a purified mixture of osteoinductive proteins (BP; Intermedics Orthopedics, Denver, CO), a serum substitute, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate. The cartilagenous fate was confirmed by 1) histological detection of sulfated proteoglycans, 2) electron microscopic detection of proteoglycan and rounded cells separated by extracellular matrix containing short, disorganized collagen fibrils, 3) morphological detection of a chondrocytes surrounded by a territorial matrix and encompassed within a distinct perichondrium, and 4) immunocytochemical detection of type II collagen and link protein. After 4 weeks in culture, mature although unmineralized cartilage was observed, as indicated by hypertrophic morphology, immunocytochemical detection of osteocalcin, and histological detection of lacunae. These conditions promote overt chondrogenesis for most of the treated cells and preclude lineage determination to the fat, muscle, and bone lineages, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartilage differentiation that we have established in vitro provides a versatile alternative to the use of chondrocyte and limb bud explant cultures. We propose this as a model system to study the factors that regulate commitment to the chondrogenic lineage, exclusion to related mesengenic pathways, and maturation during chondrogenesis. J. Cell. Biochem. 65:325–339. © 1997 Wiley-Liss, Inc.     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

The influence of oxygen and hydrostatic pressure on articular chondrocytes and adherent bone marrow cells in vitro

Tissue engineering of articular cartilage from chondrocytes or stem cells is considered to be a potential aspect in the treatment of cartilage defects. In order to optimize culture conditions the influence of low oxygen tension (5%) - single or in combination with intermittent hydrostatic pressure (HP: 30/2 min on/off loading; 0.2 MPa) - on the biosynthetic activity (sulfate and proline incorporation) of human osteoarthritic chondrocytes cultured on collagen I/III membranes was investigated. Additionally, chondrogenesis from high density or monolayer cultures of bovine adherent bone marrow cells (aBMC) with and without chondrogenic medium supplements (CM) was analyzed by RT-PCR (mRNA expression of aggrecan and collagen type II). We could show that low oxygen tension increases significantly the biosynthesis of collagen I/III membrane-associated chondrocytes and even higher under co-stimulation with HP. While there is no chondrogenesis in monolayer cultures, CM induces expression of cartilage matrix molecules in high density cultures of aBMC which is even increased under the influence of low oxygen tension. Both, low oxygen tension and HP without CM are alone not sufficient stimuli for chondrogenesis. It can be concluded that low oxygen tension and HP might be useful tools in cartilage tissue engineering and that these physico-chemical factors promote but do not induce chondrogenesis under the given conditions.     

Modification of the cell cycle of limb bud mesenchyme during in vitro cartilage differentiation

A consistent chondrogenesis takes place in micro high-density cultures derived from limb mesenchymal cells of chick embryos of stages 23-24. Flow-cytometric measurements of DNA content showed that cells in the phase of G1 or G0 made up 51% of the dispersed cell suspensions. The proportion of these cells increased to 71% by the onset of cartilage differentiation in day-2 cultures. This ratio was 84% when the voluminous matrix formation began on the 4th day of culturing. Thereafter, it increased to 90% by the 6th day, and to 93% by the 14th day. The results suggest that cartilage differentiates from G0 mesenchymal cells of the limb. In our measurements, however, the G0 phase includes all non-proliferative cell population which have identical DNA content with G1 cells. Therefore, the G0 phase contains also an increasing number of chondroblasts and chondrocytes as the chondrogenesis proceeds.     

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.     

Inhibition of limb chondrogenesis in vitro by vitamin A: alterations in cell surface characteristics

Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.     

Extracellular matrix formation by chondrocytes in monolayer culture

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

The effect of agarose and dexamethasone on the nature and production of extracellular matrix components by elastic cartilage chondrocytes

Chondrocytes isolated enzymatically from rabbit ear cartilage, were cultivated in vitro in the presence of 2% agarose or 0.1 mumol/l dexamethasone. Freshly-isolated chondrocytes suspended in either Eagle's medium or 2% agarose were auto-transplanted intramuscularly. Samples were then examined by light microscopy and transmission electron microscopy. The cells cultivated in vitro rapidly formed confluent multiple overlapping layers filled with a loose matrix consisting of single collagen fibres, proteoglycans and scarce elastic fibres. The number and maturity of the elastic fibres increased substantially after dexamethasone was added. The chondrocytes in intramuscular transplants produced a larger amount of intercellular matrix with many elastic fibres than those cultured in vitro. Addition of agarose to in vitro and in vivo systems selectively suppressed the elastin production but did not diminish the production of elastic fibre microfibrils and other matrix components. This made cultures and transplants of elastic chondrocytes resemble rather hyaline cartilage than the original tissue. It seems that the lack of elastin in the matrix does not result simply from inhibition of elastin secretion or increased elastolysis. It may be related to a reversible change of genetic expression of elastic cartilage chondrocytes under the influence of agarose.     

Genetically engineered chondrocytes overexpressing elastin improve cell retention and chondrogenesis in a three-dimensional GelMA culture system

Elastic cartilage possesses many elastic fibers and has a high degree of elasticity. However, insufficient elastic fiber production remains unsolved in elastic cartilage tissue engineering. Exogenous elastin is difficult to degrade and violates cell proliferation and migration during cartilage regeneration. Moreover, exogenous elastic fibers are difficult to assemble with endogenous extracellular matrix components. We produced genetically engineered chondrocytes overexpressing elastin to boost endogenous elastic fiber production. After identifying that genetic manipulation hardly impacted the cell viability and chondrogenesis of chondrocytes, we co-cultured genetically engineered chondrocytes with untreated chondrocytes in a three-dimensional gelatin methacryloyl (GelMA) system. In vitro study showed that the co-culture system produced more elastic fibers and increased cell retention, resulting in strengthened mechanics than the control system with untreated chondrocytes. Moreover, in vivo implantation revealed that the co-culture GelMA system greatly resisted host tissue invasion by promoting elastic fiber production and cartilage tissue regeneration compared with the control system. In summary, our study indicated that genetically engineered chondrocytes overexpressing elastin are efficient and safe for promoting elastic fiber production and cartilage regeneration in elastic cartilage tissue engineering.