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Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factor-β1 (TGF-β1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL]3. Only the combination of PDGF-BB and TGF-β1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-β1 were slowly released from the gel, as measured using their (125)I-labeled forms, and they remained significantly present in the gel at 4 days. In the context of augmenting microfracture surgery for cartilage repair, our strategy of delivering chemotactic and proanabolic growth factors in KLD may provide the necessary local stimulus to help increase defect cellularity, providing more cells to generate repair tissue.

Self-assembling peptide hydrogels were modified to deliver transforming growth factor β1 (TGF-β1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-β1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-β1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-β1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-β1 (Teth-TGF) or adsorbed TGF-β1 (Ads-TGF) were cultured in the TGF-β1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-β1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-β1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-β1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-β1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.

The objective of this study was to evaluate the effect of dynamic compression on mesenchymal stem cell (MSC) chondrogenesis. Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFbeta), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFbeta-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFbeta cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth, reaching levels in the lower portion of the hydrogel that resembled unloaded TGFbeta cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFbeta. Biosynthesis in loaded cultures was less than in unloaded TGFbeta samples. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines.