Silk fiber reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds

The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility. Both mulberry and non-mulberry silk composites showed effective swelling (25%-30%) and degradation (10%-30%) behavior, owing to their interconnected porous nature. The non-mulberry fiber-reinforced composite scaffolds showed slower degradation (∼90% mass remaining) than mulberry silk over a period of 28 days. The reinforcement of silk fibers within silk solution resulted in an increased compressive modulus and stiffness (nearly eight-fold). The biochemical analysis revealed significant increase in DNA content, sulphated glycosaminoglycan (sGAG) (∼1.5 fold) and collagen (∼1.4 fold) in reinforced composites as compared to pure solution scaffolds (p ≤ 0.01). Histological and immunohistochemical (IHC) staining corroborated enhanced deposition of sGAG and localization of collagen type II in fiber-reinforced composites. This was further substantiated by real time polymerase chain reaction studies, which indicated an up-regulation (∼1.5 fold) of cartilage-specific gene markers namely collagen type II, sox-9 and aggrecan. The minimal secretion of tumor necrosis factor-α (TNF-α) by murine macrophages further demonstrated in vitro immunocompatibility of the scaffolds. Taken together, the results signified the potential of silk fiber-reinforced composite (particularly non-mulberry, A. assamensis) scaffolds as viable alternative biomaterial for cartilage tissue engineering.