Ginsenoside Rg3-loaded liposome hydrogel promotes chondrogenic differentiation of stem cells

Abstract

BACKGROUND: Ginsenoside Rg3 has potential value in cartilage protection and repair, but its application is limited by poor water solubility, short half-life, and low bioavailability. To improve the pharmacokinetic properties of drugs, embedding drug-loaded liposomes into methylated gelatin, polysaccharide, or silk fibroin-based hydrogels has become a research hotspot in cartilage tissue engineering. OBJECTIVE: To prepare ginsenoside Rg3-loaded liposome methacrylated silk fibroin hydrogel and further analyze its effect on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells. METHODS: (1) Ginsenoside Rg3 liposomes were prepared by thin-film dispersion method. Dil-labeled liposomes were co-cultured with mouse bone marrow mesenchymal stem cells, and phalloidin staining was used to detect cellular uptake of liposomes. Methacrylated silk fibroin (SilMA) was prepared. Ginsenoside Rg3 liposomes were mixed with SilMA and crosslinked by light to prepare composite hydrogel (SilMA@Lipo-Rg3). The microstructure, mechanical properties, rheological properties, swelling properties, and drug release properties of the hydrogel were evaluated. (2) Mouse bone marrow mesenchymal stem cells were cultured with different concentrations of SilMA hydrogel extract or SilMA@Lipo-Rg3 hydrogel extract. CCK-8 assay and live/dead cell staining were used to evaluate cytocompatibility. Cells were cultured with 1/8 concentration of SilMA hydrogel extract or SilMA@Lipo-Rg3 hydrogel extract. After chondrogenic induction, qPCR was used to detect mRNA expression of collagen type II, SOX9, and aggrecan. Alcian blue and safranin O staining were used to detect the expression of proteoglycans and glycosaminoglycans. RESULTS AND CONCLUSION: (1) Phalloidin staining showed that Dil-labeled liposomes could be successfully taken up by mouse bone marrow mesenchymal stem cells. Scanning electron microscopy showed that SilMA@Lipo-Rg3 hydrogel had a loose porous network structure. Compression and rheological tests showed that the compressive stiffness of SilMA@Lipo-Rg3 hydrogel was slightly lower than that of SilMA hydrogel; there was no significant difference in swelling properties between the two hydrogels. SilMA@Lipo-Rg3 hydrogel had good sustained release performance, releasing ginsenoside Rg3 for more than 14 days. (2) CCK-8 assay and live/dead cell staining showed that SilMA and SilMA@Lipo-Rg3 hydrogels had good cytocompatibility. qPCR showed that the mRNA expression of collagen type II, SOX9, and aggrecan in the SilMA@Lipo-Rg3 group was higher than that in the SilMA group (P < 0.05). Alcian blue and safranin O staining showed that the expression of proteoglycans and glycosaminoglycans in the SilMA@Lipo-Rg3 group was higher than that in the SilMA group. These results indicate that SilMA@Lipo-Rg3 hydrogel can promote chondrogenic differentiation of mouse bone marrow mesenchymal stem cells.

1. Introduction

Cartilage defects in joints are often associated with degenerative changes in the elderly or direct/indirect trauma [1-2]. Clinical treatment for articular cartilage defects includes conservative and surgical approaches. Conservative treatment involves non-steroidal anti-inflammatory drugs and intra-articular injections, primarily aimed at relieving symptoms. However, these methods have limited efficacy in promoting cartilage regeneration. Surgical interventions, such as microfracture and autologous chondrocyte implantation, have been used, but they are invasive and may lead to fibrocartilage formation rather than hyaline cartilage. Therefore, there is a need for more effective strategies to enhance cartilage repair.

Tissue engineering approaches, combining scaffolds, cells, and growth factors, have emerged as promising alternatives. Hydrogels, due to their high water content and similarity to the native extracellular matrix, are attractive scaffolds for cartilage tissue engineering. Silk fibroin, a natural protein, has excellent biocompatibility and mechanical properties, making it a suitable candidate for hydrogel fabrication. However, its bioactivity alone may be insufficient to induce chondrogenesis. To enhance the biological function, bioactive molecules such as ginsenoside Rg3, a compound with anti-inflammatory and pro-differentiation properties, can be incorporated. Nevertheless, the poor water solubility and stability of ginsenoside Rg3 limit its direct application. Liposomes can encapsulate hydrophobic drugs, improving their stability and bioavailability. In this study, we developed a composite hydrogel by incorporating ginsenoside Rg3-loaded liposomes into methacrylated silk fibroin (SilMA) to create a sustained-release system for promoting chondrogenic differentiation of mesenchymal stem cells.