Transforming growth factor β‐3 bound with sulfate polysaccharide in synthetic extracellular matrix enhanced the biological activities for neocartilage formation in vivo

Abstract

The thermoreversible hydrogel of poly(N-isopropylacrylamide-co-acrylic acid) [p(NiPAAm-co-AAc)] was utilized in an injectable protein (growth factor) delivery system for use in tissue regeneration protocols. The transforming growth factor beta (TGF beta) proteins, which have been shown to bind to heparin, have been well established to induce chondrogenic differentiation in chondrocytes. In this study, we assessed the effects of heparin on the TGF beta-3 activities in rabbit chondrocytes embedded in thermoreversible hydrogel. The hydrogels embedded with chondrocytes and heparin-bound TGF beta-3 were injected subcutaneously into nude mice, which were monitored for up to 8 weeks after injection. P(NiPAAm-co-AAc) hydrogels with TGF beta-3 were compared with the hydrogels with heparin bound to TGF beta-3 for the assessment of the effects of heparin binding on chondrogenic differentiation. To monitor the release of growth factor via in vivo analysis, we assessed the Cy5.5-NHS-conjugated growth factors via the bioimaging method. In terms of the release of growth factors, the release of heparin-binding growth factor was slower than that of the growth factor itself. Additionally, the proliferation rate and cartilage-specific ECM production were both found to be significantly higher in the presence of heparin binding than was observed with the controls. The quantity of cartilage-associated ECM proteins was assessed via immunohistochemical staining (collagen type II) and safranin-O staining. These data indicate the potential use of heparin-binding TGF beta-3 for the reconstruction of neocartilage formation.