Deboki Naskar

The promotion of osseointegration of titanium surfaces by coating with silk protein sericin

A promising strategy to influence the osseointegration process around orthopaedic titanium implants is the immobilization of bioactive molecules. This recruits appropriate interaction between the surface and the tissue by directing cells adhesion, proliferation, differentiation and active matrix remodelling. In this study, we aimed to investigate the functionalization of metallic implant titanium with silk protein sericin. Titanium surface was immobilized with non-mulberry Antheraea mylitta sericin using glutaraldehyde as crosslinker. To analyse combinatorial effects the sericin immobilized titanium was further conjugated with integrin binding peptide sequence Arg-Gly-Asp (RGD) using ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling agents. The surface of sericin immobilized titanium was characterized biophysically. Osteoblast-like cells were cultured on sericin and sericin/RGD functionalized titanium and found to be more viable than those on pristine titanium. The enhanced adhesion, proliferation, and differentiation of osteoblast cells were observed. RT-PCR analysis showed that mRNA expressions of bone sialoprotein, osteocalcin and alkaline phosphatase were upregulated in osteoblast cells cultured on sericin and sericin/RGD immobilized titanium substrates. Additionally, no significant amount of pro-inflammatory cytokines TNF-α, IL-1β and nitric oxide production were recorded when macrophages cells and osteoblast-macrophages co culture cells were grown on sericin immobilized titanium. The findings demonstrate that the sericin immobilized titanium surfaces are potentially useful bioactive coated materials for titanium-based medical implants.

Non-mulberry silk fibroin influence osteogenesis and osteoblast-macrophage cross talk on titanium based surface

The titanium and its alloys are used as orthopedic dental implants due to their mechanical and bio-inert properties. The bare metal implants are not the ultimate answer for better osteogenesis and implant integration. Physical and chemical modifications are carried out to achieve the goal of improved adhesion and differentiation of the osteoblast. In this work, the silk fibroins from both mulberry and non-mulberry sources are used for surface modification. Silk fibroins are immobilized on titanium surface to facilitate the initial cell adhesion followed by improved cell spreading and better mineralization in order to achieve enhanced osseointegration. The immunological responses along with the effect of cytokines on osteoblast adhesion and function are investigated. The non-mulberry fibroin performs better in the context of the cell adherence and differentiation, which lead to better mineralization. The results indicate that the silk fibroin from non-mulberry source can be used for better osteogenesis on orthopedic implants.

Potential of electrospun core–shell structured gelatin–chitosan nanofibers for biomedical applications

Coaxial electrospinning is an upcoming technology that has emerged from the conventional electrospinning process in order to realize the production of nanofibers of less spinnable materials with potential applications. The present work focuses on the production of chitosan nanofibers in a benign route, using natural polymer as core template, mild solvent system and naturally derived cross-linkers. Nanofibers with chitosan as shell are fabricated by coaxial electrospinning with highly spinnable gelatin as core using aqueous acetic acid as solvent. For maintaining the biocompatibility and structural integrity of the core-shell nanofibers, cross-linking is carried out using naturally derived cross-linking agents, dextran aldehyde and sucrose aldehyde. The biological evaluation of gelatin/chitosan mat is carried out using human osteoblast like cells. The results show that the cross-linked core-shell nanofibers are excellent matrices for cell adhesion and proliferation.

Non-mulberry Silk Fibroin Biomaterial for Corneal Regeneration.

Purpose: Successful repair of a damaged corneal surface is a great challenge and may require the use of a scaffold that supports cell growth and differentiation. Amniotic membrane is currently used for this purpose, in spite of its limitations. A thin transparent silk fibroin film from non-mulberry Antheraea mylitta (Am) has been developed which offers to be a promising alternative. The silk scaffolds provide sufficient rigidity for easy handling, the scaffolds support the sprouting, migration, attachment and growth of epithelial cells and keratocytes from rat corneal explants; the cells form a cell sheet, preserve their phenotypes, express cytokeratin3 and vimentin respectively. The films also support growth of limbal stem cell evidenced by expression of ABCG2. The cell growth on the silk film and the amniotic membrane is comparable. The implanted film within the rabbit cornea remains transparent, stable. The clinical examination as well as histology shows absence of any inflammatory response or neovascularization. The corneal surface integrity is maintained; tear formation, intraocular pressure and electroretinography of implanted eyes show no adverse changes. The silk fibroin film from non-mulberry silk worms may be a worthy candidate for use as a corneal scaffold.

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.

Formulation of Biologically-Inspired Silk-Based Drug Carriers for Pulmonary Delivery Targeted for Lung Cancer.

The benefits of using silk fibroin, a major protein in silk, are widely established in many biomedical applications including tissue regeneration, bioactive coating and in vitro tissue models. The properties of silk such as biocompatibility and controlled degradation are utilized in this study to formulate for the first time as carriers for pulmonary drug delivery. Silk fibroin particles are spray dried or spray-freeze-dried to enable the delivery to the airways via dry powder inhalers. The addition of excipients such as mannitol is optimized for both the stabilization of protein during the spray-freezing process as well as for efficient dispersion using an in vitro aerosolisation impactor. Cisplatin is incorporated into the silk-based formulations with or without cross-linking, which show different release profiles. The particles show high aerosolisation performance through the measurement of in vitro lung deposition, which is at the level of commercially available dry powder inhalers. The silk-based particles are shown to be cytocompatible with A549 human lung epithelial cell line. The cytotoxicity of cisplatin is demonstrated to be enhanced when delivered using the cross-linked silk-based particles. These novel inhalable silk-based drug carriers have the potential to be used as anti-cancer drug delivery systems targeted for the lungs.

A novel in vivo platform for studying alveolar bone regeneration in rat

Alveolar bone regeneration is a significant challenge in dental implantation. Novel biomaterials and tissue-engineered constructs are under extensive development and awaiting in vivo animal tests to find clinical endpoint. Here, we establish a novel in vivo model, modifying gingivoperiosteoplasty in rat for the alveolar bone regeneration. Rat premaxillary bone defects were filled with silk scaffold or remained empty during the implantation period (up to 6 weeks), and harvested samples were analyzed by micro-computed tomography and histopathology. Empty defects showed increased but limited new bone formation with increasing implantation period. In defects implanted with silk sponge, the bone formation was significantly greater than that of empty defect, indicating an effective role of silk scaffold in the defect model. The modified premaxillary defect model in rat is simple to perform, while mimicking the clinical conditions, finding usefulness for the development of biomaterials and tissue-engineered constructs targeting alveolar bone regeneration in dental implantation.