Effects of mixing intensity on tissue-engineered cartilage

Mechanical forces regulate the structure and function of many tissues in vivo; recent results indicate that the mechanical environment can decisively influence the development of engineered tissues cultured in vitro. To investigate the effects of the hydrodynamic environment on tissue-engineered cartilage, primary bovine calf chondrocytes were seeded on fibrous polyglycolic acid meshes and cultured in spinner flasks either statically or at one of nine different turbulent mixing intensities. In medium from unmixed flasks, CO(2) accumulated and O(2) was depleted, whereas in medium from mixed flasks the concentrations of both gases approached their equilibrium values. Relative to constructs exposed to nonmixed conditions, constructs exposed to mixing contained higher fractions of collagen, synthesized and released more GAG, but contained lower fractions of GAG. Across the wide range of mixing intensities investigated, the presence or absence of mixing, but not the intensity of the mixing, was the primary determinant of the GAG and collagen content in the constructs. The all-or-none nature of these responses may provide insight into the mechanism(s) by which engineered cartilage perceives changes in its hydrodynamic environment and responds by modifying extracellular matrix production and release. 2001 John Wiley & Sons, Inc.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose‐derived stem cells: Comparison with fetal chondrocytes

This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose‐derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven‐mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF‐β1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4‐ and 6.1‐fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF‐β1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530‐fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold‐based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose‐derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393–401. © 2010 Wiley Periodicals, Inc.     

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Differential effect of ECM molecules on re-expression of cartilaginous markers in near quiescent human chondrocytes

The limited source of healthy primary chondrocytes restricts the clinical application of tissue engineering for cartilage repair. Therefore, method to maintain or restore the chondrocyte phenotype during in vitro expansion is essential. The objective of this study is to establish the beneficial effect of ECM molecules on restoring the re‐expression of cartilaginous markers in primary human chondrocytes after extensive monolayer expansion. During the course of chondrocyte serial expansion, COL2A1, SOX9, and AGN mRNA expression levels, and GAG accumulation level were reduced significantly in serially passaged cells. Exogenous type II collagen dose‐dependently elevated GAG level and induced the re‐expression of cartilaginous marker mRNAs in P7 chondrocytes. Chondroitin sulfate did not show significant effect on P7 chondrocytes, while hyaluronic acid inhibited the expression of SOX9 and AGN mRNAs. Upon treatment with type II collagen, FAK, ERK1/2, and JNK were activated via phosphorylation in P7 chondrocytes within 15 min. Furthermore, GFOGER integrin blocking peptide, MEK inhibitor and JNK inhibitor, not p38 inhibitor, significantly reduced the type II collagen‐induced GAG deposition level. Finally, in the presence of TGF‐β1 and IGF‐I, P7 chondrocytes cultured in 3D type II collagen matrix exhibited better cartilaginous features than those cells cultured in the type I collagen matrix. In conclusion, type II collagen alone can effectively restore cartilaginous features of expanded P7 human chondrocytes. It is probably mediated via the activation of FAK‐ERK1/2 and FAK‐JNK signaling pathways. The potential application of type II collagen in expanding a scarcity of healthy chondrocytes in vitro for further tissue engineering is implicated. J. Cell. Physiol. 226: 1981–1988, 2011. © 2010 Wiley‐Liss, Inc.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.     

Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.     

Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.     

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

The effect of a physiological dose of vitamin C (100 mug/ml) on goat articular cartilage chondrocytes cultured in an alginate matrix and subjected to static pressurization of 2.4 MPa was investigated. Biochemical analyses of DNA, glycosaminoglycan (GAG), collagen and protease activity were carried out in various matrix fractions, i.e. cellular matrix (CM) and further removed matrix (FRM), and in culture medium. The treatment of chondrocytes with vitamin C after static pressure increased the GAG content in both CM and FRM (P < 0.03) as compared with control or vitamin C/ static load alone. The collagen content of chondrocytes treated with vitamin C alone and vitamin C after static load also increased significantly in FRM (P < 0.003) as compared with control and static load alone. The specific activity of protease in CM and FRM decreased after vitamin C supplementation both with and without static pressure relative to control (P < 0.003). Transmission electron-microscopic images showed a mixed population of spherical and elliptical chondrocytes when vitamin C was added after static load as compared with static load alone where only elliptical cells were seen. Abundant pericellular and collagen fibrils were seen in this group of chondrocytes as compared with all other groups and the control. The results thus show that, in vitro, vitamin C supplementation of chondrocytes after static loading has the potential to reduce the morphological and biochemical degeneration of chondrocytes caused by static loading, thereby improving the cellular health and functioning of articular cartilage.     

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-β1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-β1. Expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-β1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 were synergistically increased by co-treatment with type II collagen and TGF-β1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-β1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-β1 and regulates GAG secretion in chondrocytic cells.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM-MSC) or adipose tissue (AT-MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM-MSC with bovine AC, (ii) BM-MSC from HLA-A2+ with AC from HLA-A2- donors, or (iii) human green fluorescent protein transduced BM-MSC with AC. Human BM-MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT-PCR. Coculture of NC or AC (25%) with BM-MSC or AT-MSC (75%) in pellets resulted in up to 1.6-fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM-MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2-fold) if cocultured with BM-MSC and maintained a differentiated phenotype. Chondro-induction in MSC-chondrocyte coculture is a robust process mediated by two concomitant effects: MSC-induced chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair.     

Physiological role of vitamin A in growth cartilage cells: low concentrations of retinoic acid strongly promote the proliferation of rabbit costal growth cartilage cells in culture

We have demonstrated that high concentrations of retinoic acid (RA) inhibit expression of the differentiated phenotypes of rabbit costal chondrocytes in culture [M. Takigawa et al. (1980) Proc. Natl. Acad. Sci. U.S. 77, 1481-1485]. In this study we examined the effects of low concentrations of RA on rabbit costal chondrocytes cultured in medium containing vitamin A-deficient serum. In vitamin A-deficient medium, chondrocytes isolated from growth cartilage (GC) proliferated only very slowly, and RA strongly stimulated their proliferation. This stimulatory effect was observable at a concentration of 10(-10) M RA and maximal at a concentration of 10(-8) M. RA at 10(-8) M did not change GC cells from a typical polygonal shape to fibroblast-like cells or inhibit their synthesis of type II collagen. Moreover, RA-treated cells did not synthesize type I collagen. RA inhibited glycosaminoglycan (GAG) synthesis by the cells dose-dependently, but did not change the distribution profile of proteoglycan monomers as determined by glycerol gradient centrifugation. The inhibitory action of RA on GAG synthesis was reversible: after removal of RA from the culture, the rate of GAG synthesis increased within 2 days. In contrast, resting cartilage (RC) cells proliferated well in vitamin A-deficient medium without addition of RA, and RA (10(-8) M) stimulated their proliferation only slightly. Furthermore, the inhibitory effect of RA on GAG synthesis in RC cells was much weaker than that in GC cells. These observations suggest a physiological role of RA in cartilage in stimulating the proliferation of GC cells without causing drastic change in their differentiated phenotypes.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

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.