Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage.
ABSTRACT Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair; however, it still remains a challenge to recapitulate the functional properties of native articular cartilage using only MSCs. Additionally, MSCs may exhibit a hypertrophic phenotype under chondrogenic induction, resulting in calcification after ectopic transplantation. With this in mind, the objective of this study was to assess whether the addition of chondrocytes to MSC cultures influences the properties of tissue-engineered cartilage and MSC hypertrophy when cultured in hyaluronic acid hydrogels. Mixed cell populations (human MSCs and human chondrocytes at a ratio of 4:1) were encapsulated in the hydrogels and exhibited significantly higher Young's moduli, dynamic moduli, glycosaminoglycan levels, and collagen content than did constructs seeded with only MSCs or chondrocytes. Furthermore, the deposition of collagen X, a marker of MSC hypertrophy, was significantly lower in the coculture constructs than in the constructs seeded with MSCs alone. When MSCs and chondrocytes were cultured in distinct gels, but in the same wells, there was no improvement in biomechanical and biochemical properties of the engineered tissue, implying that a close proximity is essential. This approach can be used to improve the properties and prevent calcification of engineered cartilage formed from MSC-seeded hydrogels with the addition of lower fractions of chondrocytes, leading to improved clinical outcomes.
Article: Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment.[show abstract] [hide abstract]
ABSTRACT: We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.Tissue Engineering 06/2006; 12(6):1687-97. · 4.02 Impact Factor
Article: The impact of low levels of collagen IX and pyridinoline on the mechanical properties of in vitro engineered cartilage.[show abstract] [hide abstract]
ABSTRACT: The application of in vitro engineered cartilage has become a promising approach to repair cartilage defects. Nevertheless, the poor mechanical properties of in vitro engineered cartilage limit its potential for clinical applications. Studies have shown that the extracellular matrix (ECM) components are strongly correlated with the mechanical strength of engineered cartilage, but it remains unclear which components play a key role in determining the mechanical property of engineered cartilage. To address this issue, quantitative analyses of cartilage-specific components among native cartilage, in vivo and in vitro engineered cartilages were performed, and the correlation between various ECM molecules and Young's modulus was further analyzed. The results showed that many ECM molecules, such as highly sulphated glycosaminoglycan (GAG), collagens II, IX, and pyridinoline (PYR), contributed to the mechanical strength of cartilages. Further comparison between in vitro engineered cartilage and stress-stimulated in vitro engineered cartilage, known to have stronger mechanical properties, showed that only collagen IX and PYR, but not GAG and collagen II, were the key factors determining the mechanical properties of in vitro engineered cartilage. These results indicate that in vitro environment lacks the niche for enhancing collagen crosslinking that is mediated by collagen IX and PYR during cartilage formation. Thus, the discovery provides a clue for engineering strong cartilage in vitro in the future by enhancing the levels of these two molecules.Biomaterials 12/2008; 30(5):814-21. · 7.40 Impact Factor
Article: Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage.[show abstract] [hide abstract]
ABSTRACT: A goal of cartilage tissue engineering is the production of cell-laden constructs possessing sufficient mechanical and biochemical features to enable native tissue function. This study details a systematic characterization of a serum-free (SF) culture methodology employing transient growth factor supplementation to promote robust maturation of tissue-engineered cartilage. Bovine chondrocyte agarose hydrogel constructs were cultured under free-swelling conditions in serum-containing or SF medium supplemented continuously or transiently with varying doses of transforming growth factor beta 3 (TGF-beta3). Constructs were harvested weekly or bi-weekly and assessed for mechanical and biochemical properties. Transient exposure (2 weeks) to low concentrations (2.5-5 ng/mL) of TGF-beta3 in chemically defined medium facilitated robust and highly reproducible construct maturation. Constructs receiving transient TGF-beta3 exposure achieved native tissue levels of compressive modulus (0.8 MPa) and proteoglycan content (6-7% of wet weight) after less than 2 months of in vitro culture. This maturation response was far superior to that observed after continuous growth factor supplementation or transient TGF-beta3 treatment in the presence of serum. These findings represent a significant advance in developing an ex vivo culture methodology to promote production of clinically relevant and mechanically competent tissue-engineered cartilage constructs for implantation to repair damaged articular surfaces.Tissue Engineering Part A 08/2008; 14(11):1821-34. · 4.64 Impact Factor