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The aim of this study was to investigate the effects of alginate and agarose on the response of bone marrow stromal cells (BMSCs) to chondrogenic stimuli. Rat BMSCs were expanded in monolayer culture with or without FGF-2 supplementation. Cells were then seeded in 2% alginate and agarose gels and cultured in media with or without TGF-beta1 or dexamethasone (Dex). Sulfated glycosaminoglycans (sGAGs), collagen type II, and aggrecan were expressed in all groups that received TGF-beta1 treatment during hydrogel culture. Expansion of rat BMSCs in the presence of FGF-2 increased production of sGAG in TGF-beta1-treated groups over those cultures that were treated with TGF-beta1 alone in alginate cultures. However, in agarose, cells exposed to FGF-2 during expansion produced less sGAG within TGF-beta1-supplemented groups over those cultures treated with TGF-beta1 alone. Dex was required for optimal matrix synthesis in both hydrogels, but was found to decrease cell viability in agarose constructs. These results indicate that the response of BMSCs to a chondrogenic growth factor regimen is scaffold dependent.

Summary Objective To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering. Design Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the Δ-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE). Results Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20–40 days) in PGA constructs compared to the other groups (0–20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Δ-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated Δ-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage. Conclusions The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.

Interactions between bone and cartilage formation are critical during growth and fracture healing and may influence the functional integration of osteochondral repair constructs. In this study, the ability of tissue-engineered cartilage constructs to support bone formation under controlled mechanical loading conditions was evaluated using a lapine hydraulic bone chamber model. Articular chondrocytes were seeded onto polymer disks, cultured for 4 weeks in vitro, and then transferred to empty bone chambers previously implanted into rabbit femoral metaphyses. The effects of chondrocyte viability within the implanted constructs and in vivo mechanical loading on bone formation were tested in separate experiments. After 4 weeks in vivo, biopsies from the chambers consisted of a complex composite of bone, cartilage, and fibrous tissue, with bone forming in direct apposition to the cartilage constructs. Microcomputed tomography imaging of the chamber biopsies revealed that the implantation of viable constructs nearly doubled the bone volume fraction of the chamber tissue from 0.9 to 1.6% as compared with the implantation of devitalized constructs in contralateral control chambers. The application of an intermittent cyclic mechanical load was found to increase the bone volume fraction of the chamber tissue from 0.4 to 3.6% as compared with no-load control biopsies. The results of these experiments demonstrate that tissue-engineered cartilage constructs implanted into a well-vascularized bone defect will support direct appositional bone formation and that bone formation is significantly influenced by the viability of chondrocytes within the constructs and the local mechanical environment in vivo.