Composite biomaterials as artificial bone graft materials are pushing the present frontiers of bioengineering. In this study, a biomimetic, osteoconductive tricomposite scaffold made of hydroxyapatite (HA) embedded in non-mulberry Antheraea assama (A. assama) silk fibroin fibers and its fibroin solution is explored for its osteogenic potential. Scaffolds were physico-chemically characterized for morphology, porosity, secondary structure conformation, water retention ability, biodegradability, and mechanical property. The results revealed a ∼5-fold increase in scaffold compressive modulus on addition of HA and silk fibers to liquid silk as compared to pure silk scaffolds while maintaining high scaffold porosity (∼90%) with slower degradation rates. X-ray diffraction (XRD) results confirmed deposition of HA crystals on composite scaffolds. Furthermore, the crystallite size of HA within scaffolds was strongly regulated by the intrinsic physical cues of silk fibroin. Fourier transform infrared (FTIR) spectroscopy studies indicated strong interactions between HA and silk fibroin. The fabricated tricomposite scaffolds supported enhanced cellular viability and function (ALP activity) for both MG63 osteosarcoma and human bone marrow stem cells (hBMSCs) as compared to pure silk scaffolds without fiber or HA addition. In addition, higher expression of osteogenic gene markers such as collagen I (Col-I), osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP) further substantiated the applicability of HA composite silk scaffolds for bone related applications. Immunostaining studies confirmed localization of Col-I and BSP and were in agreement with real-time gene expression results. These findings demonstrate the osteogenic potential of developed biodegradable tricomposite scaffolds with the added advantage of the affordability of its components as bone graft substitute materials.

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.