Development of a gene-activated scaffold using niosomes and minicircle DNA as a novel non-viral delivery platform to enhance cartilage repair.

Gene therapy combined with advanced biomaterial-based delivery systems represents a powerful strategy to enhance chondrogenic differentiation of mesenchymal stem cells (MSCs), enabling the development of next-generation regenerative therapies for cartilage repair. In this context, gene-activated biomaterials provide a versatile tool for spatially and temporally regulating cell fate within three-dimensional (3D) microenvironments. Here, we combine collagen type I/type II-hyaluronic acid (CI/CII-HyA) scaffold with a novel non-viral gene delivery platform based on niosomes (DP20CQ) to deliver the master chondrogenic transcription factor SOX9 using either parental (PP) or minicircle (MC) plasmids, thereby promoting chondrogenesis in MSCs. After 28 days under chondrogenic conditions, DP20CQ-based scaffolds promoted a more favourable chondrogenic-to-hypertrophic profile than gene-free or Lipofectamine (LPF)-based scaffolds while preserving metabolic activity. Sustained SOX9 overexpression was evidenced in both PP and MC niosome-based systems at the gene and protein levels. Similarly, both systems showed an upregulation of key chondrogenic markers, including aggrecan (ACAN) and collagen type II (COLII), together with the concomitant downregulation of fibrocartilage (collagen type I, COLI) and hypertrophic (collagen type X, COLX) markers, with DP20CQ/MC exhibiting the highest expression ratios. Taken together, these findings demonstrate that DP20CQ-activated biomaterials enable efficient and sustained genetic regulation in MSCs within a 3D microenvironment, promoting the formation of hyaline-like cartilage while suppressing hypertrophic differentiation. This strategy constitutes a versatile gene-activated biomaterial platform with promising potential for cartilage regeneration.