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 swelling rate, and hot water solubility of the two scaffolds were also characterized. Rabbit bone marrow mesenchymal stem cells (or human rheumatoid arthritis fibroblast-like synoviocytes) were seeded onto SF/CS/nHA scaffolds and SF/CS/nHA-ICA scaffolds, respectively. Cells cultured alone served as controls. Adhesion of the two cell types was observed under a scanning electron microscope. Cell proliferation and activity were assessed using CCK-8 assay, live/dead staining, and F-actin staining. The mRNA and protein expressions of Runx-2, osteocalcin, and type I collagen in bone marrow mesenchymal stem cells were determined using RT-qPCR and western blot assay.
RESULTS AND CONCLUSION: (1) The drug loading rate and encapsulation efficiency of icariin sustained-release microspheres were (29.38±0.04)% and (52.01±0.09)%, respectively, and icariin could be released sustainably in vitro for more than 90 days. (2) Scanning electron microscopy revealed that the interior of the two scaffolds showed a honeycomb-like porous structure with interconnected pores. There were no significant differences in pore size, porosity, water absorption expansion rate, and total hot water solubility between the two scaffolds (P > 0.05). (3) Scanning electron microscopy revealed that both cell types were tightly attached to the scaffold surface and pores, with extended pseudopodia on the surface of the SF/CS/nHA-ICA scaffold. CCK-8 assay, live/dead staining, and F-actin staining showed that the SF/CS/nHA-ICA scaffold enhanced the proliferation and viability of rabbit bone marrow mesenchymal stem cells and inhibited the proliferation and viability of human rheumatoid arthritis fibroblast-like synoviocytes compared with the control group and the SF/CS/nHA scaffold. (4) RT-qPCR and western blot assay revealed that the mRNA and protein expressions of Runx-2, osteocalcin, and type I collagen were increased in the SF/CS/nHA-ICA group compared with the control group and the SF/CS/nHA group (P < 0.05). (5) The results indicate that the icariin sustained-release microsphere three-dimensional scaffold has good cytocompatibility, in vitro osteogenesis, and anti-inflammatory effects.

Key words: icariin, sustained-release microspheres, bone tissue engineering scaffold, cytocompatibility, bone marrow mesenchymal stem cell, fibroblastic synoviocyte, biomaterial