In-situ biomimetic mineralisation of dual cross-linked silk fibroin/carboxymethyl chitosan scaffolds for bone regeneration

Abstract The structure of silk fibroin (SF) is similar to that of collagen, making it a commonly used template for mineralisation, nucleation, and growth of hydroxyapatite (HAp). However, the structure of SF has characteristics of high brittleness and poor toughness, which limits the application of pure SF as mineralisation material and needs modification. In the present work, we prepared a dual cross-linked composite scaffold (SCS) of SF and carboxymethyl chitosan (CMCS) through electrostatic attractions and ethylene glycol diglycidyl ether (EGDE)-bridged cross-links. The introduction of CMCS addressed the deficiencies of SF and provided more nucleation sites for HAp, enhancing the ability of the material to induce HAp formation, and thus better supporting cell attachment, proliferation, and differentiation. The results demonstrated successful HAp formation on mineralised scaffolds (SCS/25B and SCS/50B), with SCS/25B exhibiting optimal porosity (∼85.96%), suitable degradation rate (∼38.33%), favourable compressive strength (∼46.05 kPa), and high swelling capacity (∼1381%), meeting key requirements for porous scaffolds. Notably, SCS/25B significantly enhanced MC3T3-E1 cell proliferation, adhesion, and osteogenic differentiation (Alkaline phosphatase activity, and gene/protein expression of Runx2, OPN, OCN) compared to controls. In vivo animal studies confirmed no significant visceral toxicity in rats. Moreover, implantation of SCS/25B scaffolds for four weeks led to substantial new bone formation at the defect site. In conclusion, dual-SCS exhibits potential as a material for bone tissue engineering and provides insights into the design of SF-based biomimetic mineralisation materials.