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.

Potential of inherent RGD containing silk fibroin-poly (Є-caprolactone) nanofibrous matrix for bone tissue engineering.

The current study deals with the fabrication and characterization of blended nanofibrous scaffolds of tropical tasar silk fibroin of Antheraea mylitta and poly (Є-caprolactone) to act as an ideal scaffold for bone regeneration. The use of poly (Є-caprolactone) in osteogenesis is well-recognized. At the same time, the osteoconductive nature of the non-mulberry tasar fibroin is also established due to its internal integrin binding peptide RGD (Arg-Gly-Asp) sequences, which enhance cellular interaction and proliferation. Considering that the materials have the required and favorable properties, the blends are formed using an equal volume ratio of fibroin (2 and 4 wt%) and poly (Є-caprolactone) solution (10 wt%) to fabricate nanofibers. The nanofibers possess an average diameter of 152 ± 18 nm (2 % fibroin/PCL) and 175 ± 15 nm (4% fibroin/PCL). The results of Fourier transform infrared spectroscopy substantiates the preservation of the secondary structure of the fibroin in the blends indicating the structural stability of the neo-matrix. With an increase in the fibroin percentage, the hydrophobicity and thermal stability of the matrices as measured from melting temperature Tm (using DSC) decrease, while the mechanical strength is improved. The blended nanofibrous scaffolds are biodegradable, and support the viability and proliferation of human osteoblast-like cells as observed through scanning electron and confocal microscopes. Alkaline phosphatase assay indicates the cell proliferation and the generation of the neo-bone matrix. Taken together, these findings illustrate that the silk-poly (Є-caprolactone) blended nanofibrous scaffolds have an excellent prospect as scaffolding material in bone tissue engineering.

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.

Potential mode of protection of silkworm pupae from environmental stress by harboring the bacterial biofilm on the surfaces of silk cocoons.

The silkworm forms cocoon to protect its pupa that survives for months inside the cocoon without being affected by various environmental stresses. To understand the possible mode of pupal survival within the cocoon encasement, we investigate the cause that protects the cocoon. During the end of the spinning process, we have isolated different bacterial species from the cocoon surface. These are identified using molecular techniques and checked for their abilities to form biofilm in vitro. The bacteria are able to form biofilm either individually or in consortia. Of which, Bacillus and Erwinia species are prominent biofilm formers. Interestingly, these bacteria have the ability to form biofilm on the cocoon mimetic surface of the silk protein Sericin Hope that contains only sericin. The origin and the behavior of the bacteria lead us to hypothesize the possible role of biofilm layer on the cocoon surface, which provides protection from adverse environmental conditions.

The structure of a Plasmodium vivax Tryptophan Rich Antigen domain suggests a lipid binding function for a pan-Plasmodium multi-gene family.

Tryptophan Rich Antigens (TRAgs) are encoded by a multi-gene family found in all Plasmodium species, but are significantly expanded in P. vivax and closely related parasites. We show that multiple P. vivax TRAgs are expressed on the merozoite surface and that one, PVP01_0000100 binds red blood cells with a strong preference for reticulocytes. Using X-ray crystallography, we solved the structure of the PVP01_0000100 C-terminal tryptophan rich domain, which defines the TRAg family, revealing a three-helical bundle that is conserved across Plasmodium and has structural homology with lipid-binding BAR domains involved in membrane remodelling. Biochemical assays confirm that the PVP01_0000100 C-terminal domain has lipid binding activity with preference for sulfatide, a glycosphingolipid present in the outer leaflet of plasma membranes. Deletion of the putative orthologue in P. knowlesi, PKNH_1300500, impacts invasion in reticulocytes, suggesting a role during this essential process. Together, this work defines an emerging molecular function for the Plasmodium TRAg family.

Nonmulberry silk proteins: multipurpose ingredient in bio-functional assembly.

The emerging field of tissue engineering and regenerative medicines utilising artificial polymers is facing many problems. Despite having mechanical stability, non-toxicity and biodegradability, most of them lack cytocompatibility and biocompatibility. Natural polymers (such as collagen, hyaluronic acid, fibrin, fibroin, and others), including blends, are introduced to the field to solve some of the relevant issues. Another natural biopolymer: silkworm silk gained special attention primarily due to its specific biophysical, biochemical, and material properties, worldwide availability, and cost-effectiveness. Silk proteins, namely fibroin and sericin extracted from domesticated mulberry silkwormBombyx mori, are studied extensively in the last few decades for tissue engineering. Wild nonmulberry silkworm species, originated from India and other parts of the world, also produce silk proteins with variations in their nature and properties. Among the nonmulberry silkworm species,Antheraea mylitta(Indian Tropical Tasar),A. assamensis/A. assama(Indian Muga), andSamia ricini/Philosamia ricini(Indian Eri), along withA. pernyi(Chinese temperate Oak Tasar/Tussah) andA. yamamai(Japanese Oak Tasar) exhibit inherent tripeptide motifs of arginyl glycyl aspartic acid in their fibroin amino acid sequences, which support their candidacy as the potential biomaterials. Similarly, sericin isolated from such wild species delivers unique properties and is used as anti-apoptotic and growth-inducing factors in regenerative medicines. Other characteristics such as biodegradability, biocompatibility, and non-inflammatory nature make it suitable for tissue engineering and regenerative medicine based applications. A diverse range of matrices, including but not limited to nano-micro scale structures, nanofibres, thin films, hydrogels, and porous scaffolds, are prepared from the silk proteins (fibroins and sericins) for biomedical and tissue engineering research. This review aims to represent the progress made in medical and non-medical applications in the last couple of years and depict the present status of the investigations on Indian nonmulberry silk-based matrices as a particular reference due to its remarkable potentiality of regeneration of different types of tissues. It also discusses the future perspective in tissue engineering and regenerative medicines in the context of developing cutting-edge techniques such as 3D printing/bioprinting, microfluidics, organ-on-a-chip, and other electronics, optical and thermal property-based applications.

CCN1 expression by fibroblasts is required for bleomycin-induced skin fibrosis.

The microenvironment contributes to the excessive connective tissue deposition that characterizes fibrosis. Members of the CCN family of matricellular proteins are secreted by fibroblasts into the fibrotic microenvironment; however, the role of endogenous CCN1 in skin fibrosis is unknown. Mice harboring a fibroblast-specific deletion for CCN1 were used to assess if CCN1 contributes to dermal homeostasis, wound healing, and skin fibrosis. Mice with a fibroblast-specific CCN1 deletion showed progressive skin thinning and reduced accumulation of type I collagen; however, the overall mechanical property of skin (Young's modulus) was not significantly reduced. Real time-polymerase chain reaction analysis revealed that CCN1-deficient skin displayed reduced expression of mRNAs encoding enzymes that promote collagen stability (including prolyl-4-hydroxylase and PLOD2), although expression of COL1A1 mRNA was unaltered. CCN1-deficent skin showed reduced hydroxyproline levels. Electron microscopy revealed that collagen fibers were disorganized in CCN1-deficient skin. CCN1-deficient mice were resistant to bleomycin-induced skin fibrosis, as visualized by reduced collagen accumulation and skin thickness suggesting that deposition/accumulation of collagen is impaired in the absence of CCN1. Conversely, CCN1-deficient mice showed unaltered wound closure kinetics, suggesting de novo collagen production in response to injury did not require CCN1. In response to either wounding or bleomycin, induction of α-smooth muscle actin-positive myofibroblasts was unaffected by loss of CCN1. CCN1 protein was overexpressed by dermal fibroblasts isolated from lesional (i.e., fibrotic) areas of patients with early onset diffuse scleroderma. Thus, CCN1 expression by fibroblasts, being essential for skin fibrosis, is a viable anti-fibrotic target.

Prevention of epithelial to mesenchymal transition in colorectal carcinoma by regulation of the E-cadherin-ß-catenin-vinculin axis.

Epithelial to mesenchymal transition (EMT) is compulsory for metastatic dissemination and is stimulated by TGF-ß. Although targeting EMT has significant therapeutic potential, very few pharmacological agents have been shown to exert anti-metastatic effects. BI-69A11, a competitive Akt inhibitor, displays anti-tumor activity toward melanoma and colon carcinoma. This study provides molecular and biochemical insights into the effects of BI-69A11 on EMT in colon carcinoma cells in vitro and in vivo. BI-69A11 inhibited metastasis-associated cellular migration, invasion and adhesion by inhibiting the Akt-ß-catenin pathway. The underlying mechanism of BI-69A11-mediated inhibition of EMT included suppression of nuclear transport of ß-catenin and diminished phosphorylation of ß-catenin, which was accompanied by enhanced E-cadherin-ß-catenin complex formation at the plasma membrane. Additionally, BI-69A11 caused increased accumulation of vinculin in the plasma membrane, which fortified focal adhesion junctions leading to inhibition of metastasis. BI-69A11 downregulated activation of the TGF-ß-induced non-canonical Akt/NF-κB pathway and blocked TGF-ß-induced enhanced expression of Snail causing restoration of E-cadherin. Overall, this study enhances our understanding of the molecular mechanism of BI-69A11-induced reversal of EMT in colorectal carcinoma cells in vitro, in vivo and in TGF-ß-induced model systems.

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 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.

Dual growth factor loaded nonmulberry silk fibroin/carbon nanofiber composite 3D scaffolds for in vitro and in vivo bone regeneration.

In recent years the potential application of nanocomposite biomaterials in tissue engineering field is gaining importance because of the combined features of all the individual components. A bottom-up approach is acquired in this study to recreate the bone microenvironment. The regenerated silk protein fibroin obtained from nonmulberry tropical tasar Antheraea mylitta species is reinforced with functionalized carbon nano fiber (CNF) and the composite sponges are fabricated using facile green aqueous based method. Biophysical investigations show that the matrices are porous and simultaneously bioactive when incubated in simulated body fluid. The reinforcement of CNF influences the mechanical property of the matrices by increasing the compressive modulus up to 46.54 MPa (∼4.3 times of the control fibroin sponge) in hydrated state, which is higher than the minimum required human trabecular bone modulus (10 MPa). The composite matrices are found to be non-hemolytic as well as cytocompatible. The growth factors (BMP-2 and TGF-ß1) loaded composites show sustained release kinetics and an early attachment, growth, proliferation, and osteogenic differentiation of the osteoblasts and mesenchymal stem cells. The matrices are immunocompatible as evidenced by minimal release of pro-inflammatory cytokines both in vitro and in vivo. In order to support the in vitro study, in vivo analysis of new bone formation within the implants is performed through radiological, µ-CT, fluorochrome labeling and histological analysis, which show statistically better bone formation on growth factor loaded composite scaffolds. The study clearly shows the potential attributes of these composite matrices as an extra cellular matrix for supporting successful osseointegration process.

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 fibroin scaffolds with muscle-like elasticity support in vitro differentiation of human skeletal muscle cells.

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Non-mulberry silk fibroin grafted poly (Є-caprolactone)/nano hydroxyapatite nanofibrous scaffold for dual growth factor delivery to promote bone regeneration.

HYPOTHESIS: This study aims at developing biodegradable, mineralized, nanofibrous scaffolds for use in bone regeneration. Scaffolds are loaded with combinations of bone morphogenic protein-2 (rhBMP-2) and transforming growth factor beta (TGF-ß) and evaluated in vitro for enhancement in osteoinductivity. EXPERIMENTS: Poly(Є-caprolactone) (PCL) doped with different portions of nano-hydroxyapatite is electrospun into nanofibrous scaffolds. Non-mulberry silk fibroin (NSF) obtained from Antheraea mylitta is grafted by aminolysis onto them. Scaffolds prepared have three concentrations of nano-hydroxyapatite: 0% (NSF-PCL), 25% (NSF-PCL/n25), and 50% (NSF-PCL/n50). Growth factor loading is carried out in three different combinations, solely rhBMP-2 (BN25), solely TGF-ß (TN25) and rhBMP-2+TGF-ß (T/B N25) via carbodiimide coupling. FINDINGS: NSF-PCL/n25 showed the best results in examination of mechanical properties, bioactivity, and cell viability. Hence only NSF-PCL/n25 is selected for loading growth factors and subsequent detailed in vitro experiments using MG-63 cell-line. Both growth factors show sustain release kinetics from the matrix. The T/B N25 scaffolds support cellular activity, proliferation, and triggering of bone-associated genes' expression better and promote earlier cell differentiation. Dual growth factor loaded NSF grafted electrospun PCL/nHAp scaffolds show promise for further development into a suitable scaffold for bone tissue engineering.

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.

Metal nanoparticles triggered persistent negative photoconductivity in silk protein hydrogels.

Silk protein is a natural biopolymer with intriguing properties, which are attractive for next generation bio-integrated electronic and photonic devices. Here, we demonstrate the negative photoconductive response of Bombyx mori silk protein fibroin hydrogels, triggered by Au nanoparticles. The room temperature electrical conductivity of Au-silk hydrogels is found to be enhanced with the incorporation of Au nanoparticles over the control sample, due to the increased charge transporting networks within the hydrogel. Au-silk lateral photoconductor devices show a unique negative photoconductive response under an illumination of 325 nm, with excitation energy higher than the characteristic metal plasmon resonance band. The enhanced photoconductance yield in the hydrogels over the silk protein is attributed to the photo-oxidation of amino groups in the ß-pleated sheets of the silk around the Au nanoparticles followed by the breaking of charge transport networks. The Au-silk nanocomposite does not show any photoresponse under visible illumination because of the localization of excited charges in Au nanoparticles. The negative photoconductive response of hybrid Au-silk under UV illumination may pave the way towards the utilization of silk for future bio-photonic devices using metal nanoparticle platforms.

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.

Potential of non-mulberry silk protein fibroin blended and grafted poly(Є-caprolactone) nanofibrous matrices for in vivo bone regeneration.

An in vivo investigation is conducted to evaluate effectiveness of poly(Є-caprolactone) (PCL) nanofibrous matrices, with non-mulberry silk fibroin (NSF) (from Antheraea mylitta) inclusion, for bone tissue engineering. Inclusion is achieved by either blending NSF with PCL prior to electrospinning substrates or by grafting NSF onto electrospun PCL substrates. Proceeding from our previous in vitro results, showing that NSF grafted matrices have an edge when it comes to aiding cellular adhesion and proliferation, animal trials using rabbits are planned. As this is first in vivo trial of nanofibrous scaffolds with silk fibroin from A. mylitta, aim is to both evaluate the grafted and blended scaffolds independently and compare the method of silk fibroin introduction into the nanofibrous structures. The scaffolds are implanted at bone defect site in distal metaphysis region of the rabbits' femur. Host tissue immuno-compatibility of implants is assessed from measurements of IL-2, IL-6 and TNF-α level through 4 weeks after implantation. Barring an initial inflammatory response, IL-2, IL-6 and TNF-α levels fall back at baseline values in 2 or 4 weeks, thus confirming long term compatibility. Substantial interfacial bonding strength between grafts and host bone is evidenced from mechanical push-out test. Formation of bone tissue for both implant varieties is confirmed using histological and radiological examinations along with fluorochrome labelling and scanning electron microscopy. Significantly better bone formation is observed for NSF grafted matrices. The cumulative results from in vivo tests indicate suitability of NSF grafted PCL nanofibrous matrix as an ECM for bone repair and regrowth.

Fabrication and characterization of Eri silk fibers-based sponges for biomedical application.

Cocoon-derived semi-domesticated Eri silk fibers still lack exploitation for tissue engineering applications due to their poor solubility using conventional methods. The present work explores the ability to process cocoon fibers of non-mulberry Eri silk (Samia/Philosamia ricini) into sponges through a green approach using ionic liquid (IL)--1-buthyl-imidazolium acetate as a solvent. The formation of ß-sheet structures during Eri silk/IL gelation was acquired by exposing the Eri silk/IL gels to a saturated atmosphere composed of two different solvents: (i) isopropanol/ethanol (physical stabilization) and (ii) genipin, a natural crosslinker, dissolved in ethanol (chemical crosslinking). The sponges were then obtained by freeze-drying. This approach promotes the formation of both stable and ordered non-crosslinked Eri silk fibroin matrices. Moreover, genipin-crosslinked silk fibroin sponges presenting high height recovery capacity after compression, high swelling degree and suitable mechanical properties for tissue engineering applications were produced. The incorporation of a model drug--ibuprofen--and the corresponding release study from the loaded sponges demonstrated the potential of using these matrices as effective drug delivery systems. The assessment of the biological performance of ATDC5 chondrocyte-like cells in contact with the developed sponges showed the promotion of cell adhesion and proliferation, as well as extracellular matrix production within 2 weeks of culture. Sponges' intrinsic properties and biological findings open up their potential use for biomedical applications. STATEMENT OF SIGNIFICANCE: This work addresses the preparation and characterization of non-mulberry cocoon-derived Eri silk sponges. The insolubility of cocoons-derived non-mulberry silkworms impairs their processability and applications in the healthcare field. We used a green approach with ionic liquids to overcome the lack solubility of such silk fibers. The formation of beta-sheet structures into Eri-based sponges was physically and chemically induced. The sponges were obtained by freeze-drying. The developed structures exhibited flexibility to adapt and recover their shapes upon application and subsequent removal of load, high swelling degree, ability to load an anti-inflammatory drug and to promote its sustained release. They promoted in vitro cellular adhesion, proliferation and extracellular matrix production of a chondrocyte-like cell line, opening up their potential application for biomedical applications.