TGF-β1 fused to elastin-like polypeptides nanoparticles with kartogenin loading for mesenchymal stem cell directed cartilage differentiation and promoting <i>in vivo</i> repair

Mesenchymal stem cell (MSC) transplantation has emerged as an effective approach for treating articular cartilage damage. However, unintended hypertrophy and fibrosis of the regenerated cartilage tissue markedly undermine the therapeutic effect. Herein, a two-factor delivery system, E₆₀TK, based on the elastin-like protein ELP (VPGIG₆₀, E₆₀), co-delivering transforming growth factor β1 (TGF-β1) and kartogenin (KGN), was established to regulate stem cells from human exfoliated deciduous teeth (SHED)-directed cartilage differentiation and prevent cartilage hypertrophy and fibrosis. MTT and Alcian blue staining outcomes demonstrated that the fusion protein E₆₀T composed of E₆₀ and TGF-β1 was capable of effectively facilitating the proliferation and chondrogenic differentiation of SHED cells. Transmission electron microscope (TEM) and DLS findings revealed that the recombinant protein E₆₀T could load KGN at physiological temperature to form nanoparticles E₆₀TK, with particle sizes 241.2 ± 46.1 nm. The results of immunofluorescence and immunohistochemistry following chondrogenic differentiation suggested that E₆₀TK could efficiently induce the chondrogenic differentiation of SHED and significantly suppress the expression of type X collagen. The hyaluronic acid methacrylamide (HAMA) hydrogel loading E₆₀TK, E₆₀TK-HAMA, exhibited excellent biocompatibility, presented an interconnected porous architecture, and was completely degraded within 2 weeks <i>in vitro</i>. <i>In vivo</i> experiments indicated that the E₆₀TK-HAMA hydrogel with SHED encapsulated could effectively promote the formation of new cartilage and inhibit hypertrophy. All these results signified that E₆₀TK was an effective system for regulating the directed chondrogenic differentiation of SHED.