In vitro osteogenic and anti-inflammatory properties of icariin sustained-release microsphere three-dimensional scaffolds

Abstract

BACKGROUND: Icariin has the dual activity of promoting bone formation and inhibiting bone resorption, but its clinical application is plagued by low bioavailability, difficulty in controlling dosage, and a high risk of adverse reactions. OBJECTIVE: To prepare a three-dimensional scaffold containing icariin sustained-release microspheres and characterize their osteogenic activity in vitro. METHODS: A silk fibroin/chitosan/nanohydroxyapatite three-dimensional scaffold (SF/CS/nHA scaffold), icariin sustained-release microspheres, and a silk fibroin/chitosan/nanohydroxyapatite three-dimensional scaffold loaded with icariin sustained-release microspheres (SF/CS/nHA-ICA scaffold) were prepared. The drug loading efficiency, encapsulation efficiency, and in vitro drug release of the icariin sustained-release microspheres were characterized. The pore size, porosity, water absorption expansion rate, and hot water dissolution rate of the two scaffolds were measured. Rabbit bone marrow mesenchymal stem cells (or human rheumatoid arthritis fibroblast-like synoviocytes) were seeded on SF/CS/nHA and SF/CS/nHA-ICA scaffolds, with cells cultured alone as controls. Cell adhesion was observed by scanning electron microscopy. Cell proliferation and viability were assessed by CCK-8 assay, live/dead staining, and F-actin staining. The mRNA and protein expression of Runx-2, osteocalcin, and type I collagen in bone marrow mesenchymal stem cells were detected by RT-qPCR and western blot. RESULTS AND CONCLUSION: (1) The drug loading efficiency and encapsulation efficiency of icariin sustained-release microspheres were (29.38±0.04)% and (52.01±0.09)%, respectively, and the microspheres could sustainably release icariin for more than 90 days in vitro. (2) Scanning electron microscopy showed a honeycomb-like porous structure with interconnected pores in both scaffolds. There were no significant differences in pore size, porosity, water absorption expansion rate, or total hot water dissolution rate between the two groups (P > 0.05). (3) Scanning electron microscopy showed that both cell types adhered tightly to the scaffold surface and pores, with more extended pseudopodia on the SF/CS/nHA-ICA scaffold. CCK-8 assay, live/dead staining, and F-actin staining showed that compared with the control and SF/CS/nHA groups, the SF/CS/nHA-ICA scaffold promoted the proliferation and viability of rabbit bone marrow mesenchymal stem cells, while inhibiting the proliferation and viability of human rheumatoid arthritis fibroblast-like synoviocytes. (4) RT-qPCR and western blot showed that compared with the control and SF/CS/nHA groups, the mRNA and protein expression of Runx-2, osteocalcin, and type I collagen were increased in the SF/CS/nHA-ICA group (P < 0.05). (5) These results indicate that the icariin sustained-release microsphere three-dimensional scaffold has good cytocompatibility, and in vitro osteogenic and anti-inflammatory effects.

1. Introduction

Traditional treatments for bone defects, such as autologous or allogeneic bone grafting, have limitations including donor site morbidity, limited availability of donor bone, and potential immune rejection [1]. In osteoarthritis, synovial inflammation and degeneration are key factors. Although osteoarthritis is traditionally considered a 'non-inflammatory' arthropathy, recent studies have shown that synovitis plays an important role in its progression. Abnormal synovial proliferation and inflammation can exacerbate cartilage degeneration, and synovial cells directly and indirectly damage articular cartilage by producing enzymes and pro-inflammatory cytokines [2-4].

Bone tissue engineering offers an alternative that provides a platform for bone regeneration and is superior to traditional bone grafting. It combines biomaterials, cell therapy, growth factors, and bioreactor technologies to create and regenerate new bone tissue [5-6]. Bone marrow mesenchymal stem cells (BMSCs) have multilineage differentiation potential, immunomodulatory functions, and good tissue repair capabilities, making them a focus in tissue engineering and regenerative medicine [7]. Human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) are key effector cells in synovial hyperplasia and joint destruction. They exhibit pro-inflammatory properties by secreting inflammatory cytokines, cancer-like characteristics such as high proliferation, migration, and invasion, tissue-destructive properties by secreting proteases and promoting bone destruction, immunomodulatory effects on T cells, B cells, and dendritic cells, and can interfere with scaffold-induced osteochondral repair. Therefore, understanding the regulation of RA-FLS function and their behavior on tissue engineering scaffolds may help explore potential local therapeutic strategies [8-9].

Traditional Chinese medicine components, as emerging bioactive factors, have gained attention in bone tissue engineering. Compounds such as ginsenosides, icariin, and astragaloside IV have shown potential in promoting osteoblast proliferation and differentiation and antioxidant activity, thus promoting bone regeneration and repair. However, their application faces challenges: low bioavailability due to degradation in the digestive system and liver after oral or systemic administration; risk of adverse reactions due to off-target effects; and difficulty in dose control to achieve sufficient concentration at the defect site, often requiring high doses [10-11]. Therefore, local delivery using appropriate carriers can effectively overcome these issues.