Promita Bhattacharjee

Silk scaffolds in bone tissue engineering: An overview

Bone tissue plays multiple roles in our day-to-day functionality. The frequency of accidental bone damage and disorder is increasing worldwide. Moreover, as the world population continues to grow, the percentage of the elderly population continues to grow, which results in an increased number of bone degenerative diseases. This increased elderly population pushes the need for artificial bone implants that specifically employ biocompatible materials. A vast body of literature is available on the use of silk in bone tissue engineering. The current work presents an overview of this literature from materials and fabrication perspective. As silk is an easy-to-process biopolymer; this allows silk-based biomaterials to be molded into diverse forms and architectures, which further affects the degradability. This makes silk-based scaffolds suitable for treating a variety of bone reconstruction and regeneration objectives. Silk surfaces offer active sites that aid the mineralization and/or bonding of bioactive molecules that facilitate bone regeneration. Silk has also been blended with a variety of polymers and minerals to enhance its advantageous properties or introduce new ones. Several successful works, both in vitro and in vivo, have been reported using silk-based scaffolds to regenerate bone tissues or other parts of the skeletal system such as cartilage and ligament. A growing trend is observed toward the use of mineralized and nanofibrous scaffolds along with the development of technology that allows to control scaffold architecture, its biodegradability and the sustained releasing property of scaffolds. Further development of silk-based scaffolds for bone tissue engineering, taking them up to and beyond the stage of human trials, is hoped to be achieved in the near future through a cross-disciplinary coalition of tissue engineers, material scientists and manufacturing engineers. STATEMENT OF SIGNIFICANCE The state-of-art of silk biomaterials in bone tissue engineering, covering their wide applications as cell scaffolding matrices to micro-nano carriers for delivering bone growth factors and therapeutic molecules to diseased or damaged sites to facilitate bone regeneration, is emphasized here. The review rationalizes that the choice of silk protein as a biomaterial is not only because of its natural polymeric nature, mechanical robustness, flexibility and wide range of cell compatibility but also because of its ability to template the growth of hydroxyapatite, the chief inorganic component of bone mineral matrix, resulting in improved osteointegration. The discussion extends to the role of inorganic ions such as Si and Ca as matrix components in combination with silk to influence bone regrowth. The effect of ions or growth factor-loaded vehicle incorporation into regenerative matrix, nanotopography is also considered.

Nanofibrous nonmulberry silk/PVA scaffold for osteoinduction and osseointegration

Poly‐vinyl alcohol and nonmulberry tasar silk fibroin of Antheraea mylitta are blended to fabricate nanofibrous scaffolds for bone regeneration. Nanofibrous matrices are prepared by electrospinning the equal volume ratio blends of silk fibroin (2 and 4 wt%) with poly‐vinyl alcohol solution (10 wt%) and designated as 2SF/PVA and 4SF/PVA, respectively with average nanofiber diameters of 177 ± 13 nm (2SF/PVA) and 193 ± 17 nm (4SF/PVA). Fourier transform infrared spectroscopy confirms retention of the secondary structure of fibroin in blends indicating the structural stability of neo‐matrix. Both thermal stability and contact angle of the blends decrease with increasing fibroin percentage. Conversely, fibroin imparts mechanical stability to the blends; greater tensile strength is observed with increasing fibroin concentration. Blended scaffolds are biodegradable and support well the neo‐bone matrix synthesis by human osteoblast like cells. The findings indicate the potentiality of nanofibrous scaffolds of nonmulberry fibroin as bone scaffolding material.

Non-mulberry silk fibroin grafted poly(ε-caprolactone) nanofibrous scaffolds mineralized by electrodeposition: an optimal delivery system for growth factors to enhance bone regeneration

Mineralization of scaffolds enables them to mimic the chemistry of natural bone. Mineralizing nanofibrous scaffolds can successfully replicate both the architecture and chemical composition of bones and prove suitable for bone reconstruction. Non-mulberry silk fibroin (NSF) (from Antheraea mylitta) grafted poly(e-caprolactone) (PCL) nanofibrous scaffolds (NSF-PCL) are fabricated using electrospinning, followed by aminolysis. Electrodeposition, due to its speed and simplicity is used to deposit calcium phosphate on these scaffolds at two deposition voltages: 3 V and 5 V. The deposition of nano-hydroxyapatite (nHAp) obtained is of high quality and its topology is dependent upon the voltage of electrodeposition. Along with scaffolds of nHAp deposited on a NSF-PCL matrix at 3 V and 5 V (NSF-PCL/3V and NSF-PCL/5V respectively), the unmodified NSF-PCL matrix is used as a control. The results of mechanical characterization and certain basic cell culture using the MG-63 cell line show the merits of NSF-PCL/5V over the other two compositions. The NSF-PCL/5V scaffold is then used for detailed cell culture studies after being loaded with growth factors like bone morphogenic protein-2 (rhBMP-2) and transforming growth factor beta (TGF-β) in a 1:1 (potency) proportion. Outcomes from these studies show a clear advantage of using a combination of the growth factors over using any one of them individually. Dual growth factor loaded matrices promote more significant expression of genes related to bone growth and better facilitate early differentiation of cells. The mineralized scaffolds thus created are mechanically suitable for bone tissue engineering and in combination with growth factors significantly enhance bioactivity, proliferation and differentiation of osteoblast-like cells. The engineered scaffolds hold the potential, with further development, to serve as an optimal alternative for bone tissue engineering.

Hydroxyapatite reinforced inherent RGD containing silk fibroin composite scaffolds: Promising platform for bone tissue engineering

Replacement and repair of ectopic bone defects and traumatized bone tissues are done using porous scaffolds and composites. The prerequisites for such scaffolds include high mechanical strength, osseoconductivity and cytocompatibility. The present work is designed to address such requirements by fabricating a reinforced cytocompatible scaffold. Biocompatible silk protein fibroin collected from tropical non-mulberry tasar silkworm (Antheraea mylitta) is used to fabricate fibroin-hydroxyapatite (HAp) nanocomposite particles using chemical precipitation method. In situ reinforcement of fibroin-HAp nanocomposite and external deposition of HAp particles on fibroin scaffold is carried out for comparative evaluations of bio-physical and biochemical characteristics. HAp deposited fibroin scaffolds provide greater mechanical strength and cytocompatibility, when compared with fibroin-HAp nanoparticles reinforced fibroin scaffolds. Minimal immune responses of both types of composite scaffolds are observed using osteoblast-macrophage co-culture model. Nanocomposite reinforced fibroin scaffold can be tailored further to accommodate different requirements depending on bone type or bone regeneration period.

Animal trial on zinc doped hydroxyapatite: A case study

Abstract Calcium hydroxyapatite (HAp) has widely been used as bone substitute due to its good biocompatibility and bioactivity. In the present work, hydroxyapatite was doped with zinc (Zn) to improve its bioactivity. The study reports the technique to synthesize Zn-doped HAp powder using a simple, economic route and the influence of this dopant on the physical, mechanical and biological properties of the HAp. Porous blocks were prepared by sintering at 1150 °C and the sintered samples were characterized using XRD and FTIR. In vitro bioresorption behavior of the sintered blocks was assessed in simulated body fluid (SBF) maintained in a dynamic state. The in vivo study was exclusively conducted to evaluate healing of surgically created defects on the tibia of adult New Zealand rabbit after implantation of HAp. Local inflammatory reaction and healing of wound, radiological investigations, histological and SEM studies, oxytetracycline labeling and mechanical push-out test were performed up to 60 days post-operatively. It was observed that Zn substituted HAp showed better osteointegration than undoped HAp. Radiology revealed progressively less contrast between implant and surrounding bone. New bone formation in Zn-doped HAp was more prompt. Mechanical push-out test showed high interfacial strength (nearly 2.5 times) between host bone and doped implant.

Carbon Nanofiber Reinforced Nonmulberry Silk Protein Fibroin Nanobiocomposite for Tissue Engineering Applications

Natural silk protein fibroin based biomaterial are exploited extensively in tissue engineering due to their aqueous preparation, slow biodegradability, mechanical stability, low immunogenicity, dielectric properties, tunable properties, sufficient and easy availability. Carbon nanofibers are reported for their conductivity, mechanical strength and as delivery vehicle of small molecules. Limited evidence about their cytocompatibility and their poor dispersibility are the key issues for them to be used as successful biomaterials. In this study, carbon nanofiber is functionalized and dispersed using the green aqueous-based method within the regenerated nonmulberry (tropical tasar: Antheraea mylitta) silk fibroin (AmF), which contains inherent - R-G-D- sequences. Carbon nanofiber (CNF) reinforced silk films are fabricated using solvent evaporation technique. Different biophysical characterizations and cytocompatibility of the composite matrices are assessed. The investigations show that the presence of the nanofiber greatly influence the property of the composite films in terms of excellent conductivity (up to 6.4 × 10-6 Mho cm, which is 3 orders of magnitude of pure AmF matrix), and superior tensile modulus (up to 1423 MPa, which is 12.5 times more elastic than AmF matrix). The composite matrices (composed of up to 1 mg of CNF per mL of 2% AmF) also support better fibroblast cell growth and proliferation. The fibroin-carbon nanofiber matrices can lead to a novel multifunctional biomaterial platform, which will support conductive as well as load bearing tissue (such as, muscle, bone, and nerve tissue) regenerations.

Silk fibroin-Thelebolan matrix: A promising chemopreventive scaffold for soft tissue cancer

Research of improved functional bio-mimetic matrix for regenerative medicine is currently one of the rapidly growing fields in tissue engineering and medical sciences. This study reports a novel bio-polymeric matrix, which is fabricated using silk protein fibroin from Bombyx mori silkworm and fungal exopolysaccharide Thelebolan from Antarctic fungus Thelebolus sp. IITKGP-BT12 by solvent evaporation and freeze drying method. Natural cross linker genipin is used to imprison the Thelebolan within the fibroin network. Different cross-linked and non-cross-linked fibroin/Thelebolan matrices are fabricated and biophysically characterized. Cross-linked thin films show robustness, good mechanical strength and high temperature stability in comparison to non-cross-linked and pure matrices. The 3D sponge matrices demonstrate good cytocompatibility. Interestingly, sustained release of the Thelebolan from the cross-linked matrices induce apoptosis in colon cancer cell line (HT-29) in time dependent manner while it is nontoxic to the normal fibroblast cells (L929).The findings indicate that the cross-linked fibroin/Thelebolan matrices can be used as potential topical chemopreventive scaffold for preclusion of soft tissue carcinoma.

Silk-based matrices for bone tissue engineering applications

Abstract A growing world population with rapidly rising fractions of elderly and traumatic bone fracture cases makes bone tissue engineering (BTE) a necessity of the current times. Developing low-cost and biocompatible scaffolds using bioderived materials could be the logical choice for bone tissue repair. Silk is a biopolymer with several characteristics, including excellent biocompatibility and mechanical strength that makes it a potential candidate for various tissue engineering applications. There exists a vast body of literature regarding the use of silk in BTE. Several successful works have reported use of silk scaffolds for bone repair and regeneration. These works involve trials both in vitro and in vivo. A growing trend is observed towards designing mineralized nanofibrous and composite scaffolds. This chapter presents an overview of the field, from the perspective of materials and fabrication.