Controlled Delivery of Transforming Growth Factor β1 by Self-Assembling Peptide Hydrogels Induces Chondrogenesis of Bone Marrow Stromal Cells and Modulates Smad2/3 Signaling

Abstract

Self-assembling peptide hydrogels were modified to deliver transforming growth factor b1 (TGF-b1) 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-b1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-b1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-b1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-b1 (Teth-TGF) or adsorbed TGF-b1 (Ads-TGF) were cultured in the TGF-b1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-b1 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-b1 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-b1 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-b1 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.

Hydrogels for the Repair of Articular Cartilage Defects

“…The inclusion of TGFb1 to agarose, peptide, and collagen hydrogels was sufficient to induce chondrogenesis of encapsulated MSCs, 225,226 which was further enhanced by including dexamethasone. 226 The delivery of TGFb3 and hyaluronic acid alone or in combination enhanced chondrogenesis of MSCs in PEG hydrogels; TGFb3 was required for proteoglycan production and hyaluronic acid reduced the production of type I collagen.…”

Growth Factor Delivery Through Self-assembling Peptide Scaffolds

“…The use of HB-IGF-1 with the KLD peptide system could be enabled by mixing in heparin, heparan sulfate, or the heparan sulfate proteoglycan, perlecan, with KLD [18,19,51]. Although adsorption of TGF-b1 to KLD effectively increased proteoglycan production in our study and a previous study [26], changing the degradation rates of KLD by changing the concentration or adding crosslinks may offer a way of improving delivery.…”

“…RAD reportedly supports growth factor delivery: PDGF-BB and other proteins have been adsorbed to RAD [17,27], SDF-1 was inserted directly onto the RAD peptide sequence [41], and IGF-1 was tethered to RAD through biotin-streptavidin-biotin bonds [6] successfully stimulating cardiomyocytes in vivo. Previous work established that TGF-b1 adsorbed to KLD stimulated chondrogenesis of BMSCs [26].…”

Delivering Heparin-Binding Insulin-Like Growth Factor 1 with Self-Assembling Peptide Hydrogels

“…To determine a quantitative release profile, 14 C-IGF-1 or 14 C-HB-IGF-1 was mixed into unassembled RAD peptide solution at a concentration of 615 nM. Thirty microliters of PBS with PSA was layered on top of 50 mL of HB-IGF-1-or IGF-1-functionalized RAD to initiate hydrogel selfassembly.…”