Although BMSC-based therapy is one of the most front-line technologies for cartilage repair, it is still a big challenge to attain ideal niches for BMSC chondrogenic differentiation. In this study, we developed hyaluronate and chondroitin sulfate derivatives to prepare covalently crosslinked polysaccharide hydrogels. Based on these binary hydrogels, collagen was added to prepare ternary hybrid hydrogels and its effect on encapsulated BMSCs was studied. After culturing with different cell densities in vitro without the addition of growth factors for 3 weeks, the chondrogenesis of BMSCs was evaluated by CLSM, mechanical testing, histological staining, immunohistochemical staining and gene expression. The results indicated that BMSCs in high cell density (50 million per mL) cell-laden constructs had a more obvious chondrogenic phenotype than those in low cell density ones (5 million per mL). However, the components of hydrogels had a significant influence on chondrogenic differentiation. With the addition of collagen, the BMSCs in ternary hybrid hydrogels showed more significant chondrogenesis, possessing with more amounts of secreted glycosaminoglycans (GAGs) and type II collagen deposition, higher mechanical properties and chondrogenic gene expression over 3 weeks of culture in vitro. It can be concluded that the bioactive collagen is beneficial to the chondrogenesis of BMSCs. This hybrid hydrogels deserve further studies to have a prospective application in tissue engineering for cartilage defect repair.

Although BMSC-based therapy is one of the most front-line technologies for cartilage repair, it is still a big challenge to attain ideal niches for BMSC chondrogenic differentiation. In this study, we developed hyaluronate and chondroitin sulfate derivatives to prepare covalently crosslinked polysaccharide hydrogels. Based on these binary hydrogels, collagen was added to prepare ternary hybrid hydrogels and its effect on encapsulated BMSCs was studied. After culturing with different cell densities in vitro without the addition of growth factors for 3 weeks, the chondrogenesis of BMSCs was evaluated by CLSM, mechanical testing, histological staining, immunohistochemical staining and gene expression. The results indicated that BMSCs in high cell density (50 million per mL) cell-laden constructs had a more obvious chondrogenic phenotype than those in low cell density ones (5 million per mL). However, the components of hydrogels had a significant influence on chondrogenic differentiation. With the addition of collagen, the BMSCs in ternary hybrid hydrogels showed more significant chondrogenesis, possessing with more amounts of secreted glycosaminoglycans (GAGs) and type II collagen deposition, higher mechanical properties and chondrogenic gene expression over 3 weeks of culture in vitro. It can be concluded that the bioactive collagen is beneficial to the chondrogenesis of BMSCs. This hybrid hydrogels deserve further studies to have a prospective application in tissue engineering for cartilage defect repair.

Mesenchymal stem cells (MSCs) had been increasingly regarded as a potent cell source for cartilage repair. However, due to the instability of MSC-derived chondrocyte phenotype and ossification of the synthesised cartilage matrix, regenerating a stable cartilage tissue by MSCs is still challenging. The fate of chondrogenesis from MSCs is regulated by their local microenvironment, which is of vital importance to the cell behaviours, chondrogenic phenotype and matrix synthesis. In this study, we fabricated cartilage-like tissues by the chondrogenesis of MSC in three different microenvironments, including cell pellets, collagen hydrogel bulk (CHB) and collagen hydrogel microspheres (CHMs) in vitro. After 15 days in culture, the cell number was increased to 472.6% in CHMs, compared to a 58.6% decrease in CHB and a 46.6% decrease in pellets; resulting in a 230% increase in CHM size, but a 36.8% decrease in CHB and only a 20.1% increase in pellets. Histological staining demonstrated a more intensive but less homogeneous glycosaminoglycan (GAG) pattern in pellets than in CHMs. The outer area of CHB showed a stronger GAG staining than its inner area from day 5 to day 15, but the staining was weaker than that in both pellets and CHMs. The PCR results showed that CHMs achieved a significantly higher chondrogenic gene (AGG, COL2A1, SOX9) expression and a lower hypertrophic gene (COL10A1) expression than pellets and CHB, suggesting a better chondrogenic differentiation potential with a more stable phenotype in CHMs. In summary, this study highlights the advantages of CHM microenvironments over those of CHB and pellets by a better mimicking of the natural MSC proliferation process and enhancing mass exchange in vitro. The CHM culture demonstrated potential to fabricate stable cartilage-like tissue in MSC based cartilage tissue regeneration.

•A novel hyaluronic acid modification method was developed.•DS of maleated hyaluronan (MaHA) is higher than methacrylated hyaluronan's (MeHA).•The hydrogels of MaHA have higher compressive storage moduli than those of MeHA.•The crosslinking density and hydrophilicity of introduced groups affect the swelling.A series of maleated hyaluronan (MaHA) are developed by modification with maleic anhydride. The degrees of substitution (DS) of MaHA vary between 7% and 75%. The DS of MaHA is both higher and wider than methacrylated HA derivatives (MeHA) reported in the literature. MaHA hydrogels are then prepared by photopolymerization and their dynamic mechanical and swelling properties of the hydrogels are investigated. The results showed that MaHA hydrogels with moderate DS (25%, 50% and 65%) have higher storage modulus and lower equilibrium swelling ratios than those with either low or high DS (7%, 15% and 75%). Theoretical analyses also suggest a similar pattern among hydrogels with different DS. The results confirm that the increased cross-linking density enhances the strength of hydrogels. Meanwhile, the hydrophilicity of introduced groups during modification and the degree of incomplete crosslinking reaction might have negative impact on the mechanical and swelling properties of MaHA hydrogels.

Cell niche, which is considered to be critical to the proliferation and differentiation of cells, is one of the most important aspects for the design and development of ideal scaffolds in tissue engineering. The mass transfer property of the scaffold affects the nutrient supply and exchange of the other substances. In this study, we prepared collagen hydrogels in the form of microspheres (CHMs) and bulk (CHB) to investigate the mass exchange differences and their influence on embedded chondrocytes. CHMs were developed by the emulsion method, which was efficient to load cells. Bovine serum albumin (BSA) was used as a diffusion model in the CHMs and CHB to evaluate the transport property of the hydrogels and the release kinetics. During the 4 week in vitro culture process, the contraction of the hydrogels, the cell viability and morphology, and the DNA and glycosaminoglycan (GAG) content were monitored at different intervals. The results suggested that the CHMs showed an obvious superiority in the transfer property over the CHB, leading to better maintenance of the chondrocyte phenotype in CHMs at the early stage of the in vitro culture. Histological analyses indicated that lots of lacunae and homogeneous positive GAG staining appeared in the CHMs from day 7. In contrast, only a few lacunae and obscure GAG staining were found in the outer area of the CHB after day 21. Without enough nutrients, the chondrocytes in the inner area of the CHB had little secreted matrix. Based on the presented CHM system, a further developed construct is suggested as a promising alternative toward the clinical application of engineered cartilaginous tissue.

Although BMSC-based therapy is one of the most front-line technologies for cartilage repair, it is still a big challenge to attain ideal niches for BMSC chondrogenic differentiation. In this study, we developed hyaluronate and chondroitin sulfate derivatives to prepare covalently crosslinked polysaccharide hydrogels. Based on these binary hydrogels, collagen was added to prepare ternary hybrid hydrogels and its effect on encapsulated BMSCs was studied. After culturing with different cell densities in vitro without the addition of growth factors for 3 weeks, the chondrogenesis of BMSCs was evaluated by CLSM, mechanical testing, histological staining, immunohistochemical staining and gene expression. The results indicated that BMSCs in high cell density (50 million per mL) cell-laden constructs had a more obvious chondrogenic phenotype than those in low cell density ones (5 million per mL). However, the components of hydrogels had a significant influence on chondrogenic differentiation. With the addition of collagen, the BMSCs in ternary hybrid hydrogels showed more significant chondrogenesis, possessing with more amounts of secreted glycosaminoglycans (GAGs) and type II collagen deposition, higher mechanical properties and chondrogenic gene expression over 3 weeks of culture in vitro. It can be concluded that the bioactive collagen is beneficial to the chondrogenesis of BMSCs. This hybrid hydrogels deserve further studies to have a prospective application in tissue engineering for cartilage defect repair.

Although BMSC-based therapy is one of the most front-line technologies for cartilage repair, it is still a big challenge to attain ideal niches for BMSC chondrogenic differentiation. In this study, we developed hyaluronate and chondroitin sulfate derivatives to prepare covalently crosslinked polysaccharide hydrogels. Based on these binary hydrogels, collagen was added to prepare ternary hybrid hydrogels and its effect on encapsulated BMSCs was studied. After culturing with different cell densities in vitro without the addition of growth factors for 3 weeks, the chondrogenesis of BMSCs was evaluated by CLSM, mechanical testing, histological staining, immunohistochemical staining and gene expression. The results indicated that BMSCs in high cell density (50 million per mL) cell-laden constructs had a more obvious chondrogenic phenotype than those in low cell density ones (5 million per mL). However, the components of hydrogels had a significant influence on chondrogenic differentiation. With the addition of collagen, the BMSCs in ternary hybrid hydrogels showed more significant chondrogenesis, possessing with more amounts of secreted glycosaminoglycans (GAGs) and type II collagen deposition, higher mechanical properties and chondrogenic gene expression over 3 weeks of culture in vitro. It can be concluded that the bioactive collagen is beneficial to the chondrogenesis of BMSCs. This hybrid hydrogels deserve further studies to have a prospective application in tissue engineering for cartilage defect repair.