Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels.

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.

Enhanced MSC chondrogenesis following delivery of TGF-ß3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo.

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-ß) superfamily are a key mediator of MSC chondrogenesis. While TGF-ß mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-ßs in vivo. Here, we investigated the co-encapsulation of TGF-ß3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-ß3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-ß3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-ß3 continuously in culture media, whereas constructs with TGF-ß3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-ß3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-ß3 or with TGF-ß3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-ß3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-ß3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.

Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage.

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.