Development of Novel Silk Fibroin/Carboxymethyl Cellulose Based Electrospun Nanofibrous Scaffolds for Bone Tissue Engineering Application

Abstract

Bone Tissue Engineering (TE) has been evolved as a promising mean to repair and regenerate defect and/or diseased bone tissues through the development of artificial extracellular matrix made from biopolymers. Silk fibroin based biomaterials are considered ideal for developing scaffolds for TE applications. The present research aims to develop novel biomimetic silk fibroin (SF) and carboxymethyl cellulose (CMC) based nanofibrous scaffold by free liquid surface electrospinning method. Randomly oriented electrospun nanofibrous scaffolds were developed from SF/CMC blends. Among the different blend ratios, 98:2 w/w of SF/CMC2 is the optimal achieving a set of superior scaffold properties in comparison to pure SF scaffold. The scaffolds were characterized for morphology (SEM, AFM), structural (XRD, FT-IR), surface property (% water uptake and contact angle measurement) and mechanical strength. The average fiber diameter was obtained as 227.8 ± 87 nm. The scaffold exhibited higher water uptake capacity, hydrophilicity and bioactivity showing uniform nucleation of Ca/P over the surface with controlled particle size of ≤ 100 nm than the pure bombyx mori SF scaffolds. Though the tensile strength of pure SF (12.7 ± 1.5 MPa) was slightly reduced by the addition of CMC (10.54 ± 1.3 MPa), the scaffold still possess sufficient strength to support many types of bone tissue regeneration. In vitro cell culture study has confirmed the cell supportive property of the scaffold as evident from cell attachment, cell proliferation, cell penetration and cellular metabolic activity of hMSCs which was derived from umbilical cord blood over the scaffold. The developed calcified SF/CMC2 blend scaffold possess good osteogenic property as confirmed by ALP activity, biomineralization ability, GAG secretion, osteocalcin, RUN X2 and collagen type1 expression. The osteogenic potential of SF/CMC scaffold was further enhanced by incorporating nano-bioglass thereby SF/CMC/nBG composite scaffold was developed. The surface roughness of SF/CMC/nBG (5 - 20 wt%) scaffolds was linearly increased with nano-bioglass content. Among the various concentration of nBG, SF/CMC loaded with 10%nBG shows optimal tensile strength of 7.591 ± 1.23 MPa and tensile strain at break of 9.62 ± 0.85 %. The ALP activity of hMSCs on SF/CMC/10nBG was significantly (p˂0.05) higher than SF/CMC2 throughout the culture period. The OCN expression on SF/CMC/10%nBG was observed to be 4.1 fold higher than SF/CMC2 and 2.3 fold higher than calcified SF/CMC2 nanofibrous scaffold. Hence, SF/CMC/10%nBG composite scaffold is proven to provide better osteogenic platform for hMSCs. An effort has further been given to improve the angiogenic property of SF/CMC/nBG composite scaffolds by incorporating Cu2+ ion. Among the scaffolds with varied compositions, SF/CMC/Cu1-5%nBG exhibited superior osteogenic and angiogenic property. Though a slightly higher OCN expression was observed over SF/CMC/Cu0.5-5%nBG composite scaffold than SF/CMC/Cu1-5%nBG, the difference in the expression was not statistically significant. However, SF/CMC/Cu1-5%nBG shows higher VEGF expression representing its superior angiogenic property than SF/CMC/Cu0.5-5%nBG scaffold. Thus, copper doped nano bioglass incorporated SF/CMC/Cu1-5%nBG composite nanofibrous matrix was proven to be a potential artificial extracellular matrix with enhanced bioactivity, osteogenic and angiogenic properties which might be a promising scaffold for future bone tissue regeneration.