Optimization and evaluation of oxygen-plasma-modified, aligned, poly (Є-caprolactone) and silk fibroin nanofibrous scaffold for corneal stromal regeneration.

The shortage of human donor corneas for transplantation necessitates the exploration of tissue engineering approaches to develop corneal substitutes. However, these substitutes must possess the necessary strength, transparency, and ability to regulate cell behaviour before they can be used in patients. In this study, we investigated the effectiveness of an oxygen plasma surface-modified poly-ε-caprolactone (PCL) combined with silk fibroin (SF) nanofibrous scaffold for corneal stromal regeneration. To fabricate the electrospun scaffolds, PCL and SF blends were used on a rotating mandrel. The optimization of the blend aimed to replicate the structural and functional properties of the human cornea, focusing on nanofibre alignment, mechanical characteristics, and in vitro cytocompatibility with human corneal stromal keratocytes. Surface modification of the scaffold resulted in improved transparency and enhanced cell interaction. Based on the evaluation, a composite nanofibrous scaffold with a 1:1 blend of PCL and SF was selected for a more comprehensive analysis. The biological response of keratocytes to the scaffold was assessed through cellular adhesion, proliferation, cytoskeletal organization, gene expression, and immunocytochemical staining. The scaffold facilitated the adhesion of corneal stromal cells, supporting cell proliferation, maintaining normal cytoskeletal organization, and promoting increased expression of genes associated with healthy corneal stromal keratocytes. These findings highlight the potential of a surface-modified PCL/SF blend (1:1) as a promising scaffolding material for corneal stromal regeneration. The developed scaffold not only demonstrated favourable biological interactions with corneal stromal cells but also exhibited characteristics aligned with the requirements for successful corneal tissue engineering. Further research and refinement of these constructs could lead to significant advancements in addressing the shortage of corneas for transplantation, ultimately improving the treatment outcomes for patients in need.

Impact of Propeptide Cleavage on the Stability and Activity of a Streptococcal Immunomodulatory C5a Peptidase for Biopharmaceutical Development.

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

Silk fibroin based interpenetrating network hydrogel for corneal stromal regeneration.

There is a need to develop tissue engineering based approaches to address the shortage of donor corneas worldwide for transplantation. To do this a novel approach to fabricate three-dimensional hydrogels using free-radical polymerization was investigated to generate constructs for corneal stromal tissue regeneration. Different ratios of silk fibroin (SF) to polyacrylamide (PA) were used to fabricate semi-interpenetrating hydrogels. Scanning electron micrograph displayed the interconnectivity of pores within the fabricated hydrogels. Pore sizes ranged from 25 to 66 µm. Scaffolds with increasing concentration of SF had enhanced ß-sheet structure (verified by Fourier transform infrared spectroscopy). The biological response of human corneal stromal cells to these hydrogels was examined using cellular adhesion, proliferation, cytoskeleton organization, gene expression and immunocytochemical analysis. The fabricated hydrogels possess rapid gelation (∼3 min) at 37 °C, 84 % porosity facilitating keratocyte migration during healing, improved cellular adhesion and no cytotoxicity, indicating their efficiency for in-situ corneal tissue regeneration. Presence of SF in semi-interpenetrating network hydrogel enhanced cellular proliferation, elevated GAG deposition, and increased expression of keratocyte genes, normally associated with healthy corneal stromal tissue. This study acts as an initial step towards fabricating SF based semi-interpenetrating network hydrogels for developing clinically applicable ocular implants.

Influence of spiral topographies on human limbal-derived immortalized corneal epithelial cells.

Cells migrate from the limbus to the corneal epithelium following a centripetal pathway. Corneal epithelial cells tend to orientate in spiral or vortex patterns. However, when cultured in-vitro, limbal derived corneal epithelia do not tend to align or migrate in a spiral pattern. Here, we used soft lithography to create silk fibroin substrates with spiral topographies that direct the human limbal-derived immortalized corneal epithelial cells (hTCEpi) to form a spiral orientation. The impact of this topography on the cells was then characterized. The spiral patterns affected cytoskeletal organization, cell spreading, and nuclei shapes. Spiral width and numbers had a significant impact on proliferation of cells, their focal adhesion, their chromatin condensation, and number of actin filaments. Immunocytochemical staining showed that the spiral pattern enhanced the expression of markers associated with limbal stem cells. The current work illustrates micro spiral patterns can serve to control the nature of limbal derived epithelial cells by providing relevant biophysical cues.

Biomedical applications of silk and its role for intervertebral disc repair.

Intervertebral disc (IVD) degeneration (IDD) is the main contributor to chronic low back pain. To date, the present therapies mainly focus on treating the symptoms caused by IDD rather than addressing the problem itself. For this reason, researchers have searched for a suitable biomaterial to repair and/or regenerate the IVD. A promising candidate to fill this gap is silk, which has already been used as a biomaterial for many years. Therefore, this review aims first to elaborate on the different origins from which silk is harvested, the individual composition, and the characteristics of each silk type. Another goal is to enlighten why silk is so suitable as a biomaterial, discuss its functionalization, and how it could be used for tissue engineering purposes. The second part of this review aims to provide an overview of preclinical studies using silk-based biomaterials to repair the inner region of the IVD, the nucleus pulposus (NP), and the IVD's outer area, the annulus fibrosus (AF). Since the NP and the AF differ fundamentally in their structure, different therapeutic approaches are required. Consequently, silk-containing hydrogels have been used mainly to repair the NP, and silk-based scaffolds have been used for the AF. Although most preclinical studies have shown promising results in IVD-related repair and regeneration, their clinical transition is yet to come.

Role of calcium phosphate and bioactive glass coating on in vivo bone healing of new Mg-Zn-Ca implant.

Present investigation focuses on development and detailed characterization of a new Mg alloy sample (BM) with and without coating of hydroxyapatite (BMH) and bioactive glass (BMG) by air plasma spray method. After detailed mechano-physico-chemical characterization of powders and coated samples, electrochemical corrosion and SBF immersion tests were carried out. Detailed in vitro characterizations for cell viability were undertaken using MG-63 cell line followed by in vivo tests in rabbit model for studying bone healing up to 60 days. Starting current density increases from BM to BMH to BMG indicating highest resistance towards corrosion in case of BMG samples, however BMH also showed highest icorr value suggesting slowest rate of corrosion than BM and BMG samples. Dissolution of calcium ion in case of BMH and BMG control formation of apatite phases on surface. Ca2+ ions of coatings and from SBF solution underwent reduction reaction simultaneously with conversion of Mg to MgCl2 releasing OH- in the solution, which increases pH. Viability and propagation of human osteoblast-like cells was verified using confocal microscopy observations and from expression of bone specific genes. Alkaline phosphatase assay and ARS staining indicate cell proliferation and production of neo-osseous tissue matrix. In vivo, based on histology of heart, kidney and liver, and immune response of IL-2, IL-6 and TNFα, all the materials show no adverse effects in body system. The bone creation was observed to be more for BMH. Although both BMH and BMG show rays of possibilities in early new bone formation and tough bone-implant bonding at interface as compared to bare Mg alloy, however, BMG showed better well-sprayed coating covering on substrate and resistance against corrosion prior implanting in vivo. Also, better apatite formation on this sample makes it more favourable implant.

Significance of Crosslinking Approaches in the Development of Next Generation Hydrogels for Corneal Tissue Engineering.

Medical conditions such as trachoma, keratoconus and Fuchs endothelial dystrophy can damage the cornea, leading to visual deterioration and blindness and necessitating a cornea transplant. Due to the shortage of donor corneas, hydrogels have been investigated as potential corneal replacements. A key factor that influences the physical and biochemical properties of these hydrogels is how they are crosslinked. In this paper, an overview is provided of different crosslinking techniques and crosslinking chemical additives that have been applied to hydrogels for the purposes of corneal tissue engineering, drug delivery or corneal repair. Factors that influence the success of a crosslinker are considered that include material composition, dosage, fabrication method, immunogenicity and toxicity. Different crosslinking techniques that have been used to develop injectable hydrogels for corneal regeneration are summarized. The limitations and future prospects of crosslinking strategies for use in corneal tissue engineering are discussed. It is demonstrated that the choice of crosslinking technique has a significant influence on the biocompatibility, mechanical properties and chemical structure of hydrogels that may be suitable for corneal tissue engineering and regenerative applications.

Fabrication and Biocompatibility of Electroconductive Silk Fibroin/PEDOT: PSS Composites for Corneal Epithelial Regeneration.

The aim of this study was to develop matrices that can support human corneal epithelial cells and innervation by incorporating a conducting polymer, poly(3,4-ethylenedioxythiophene) poly(styrene sulfonate) (PEDOT:PSS), into silk fibroin (SF). Polyvinyl alcohol (PVA) was used as a crosslinking agent to enhance the mechanical properties of the matrices. The impact of PEDOT:PSS on the materials' physical properties and cellular responses was examined. The electrical impedance of matrices decreased with increasing concentration of PEDOT:PSS suggesting improved electroconductivity. However, light transmittance also decreased with increasing PEDOT:PSS. Young's modulus was unaffected by PEDOT:PSS but was increased by PVA. The viability of corneal epithelial cell on the matrices was unaffected by the incorporation of PEDOT:PSS except at the highest concentration tested 0.3% (w/v), which led to a cytotoxic response. These findings suggest that SF/PEDOT:PSS with a PEDOT:PSS concentration of 0.1-0.2% would be a suitable biomaterial for epithelium regeneration.

Influence of micropatterned substrates on keratocyte phenotype.

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.

Development of nano-porous hydroxyapatite coated e-glass for potential bone-tissue engineering application: An in vitro approach.

To reconstruct the defects caused by craniectomies autologous, bone grafting was usually used, but they failed most commonly due to bone resorption, infections and donor-site morbidity. In the present investigation, an effort has been made for the first time to check the feasibility and advantage of using hydroxyapatite (HAp) coated e-glass as component of bone implants. Sol-gel synthesized coatings were found to be purely hydroxyapatite from XRD with graded and interconnected pores all over the surface observable in TEM. The interconnected porous nature of ceramics are found to increase bioactivity by acting to up-regulate the process of osseointegration through enhanced nutrient transfer and induction of angiogenesis. From TEM studies and nano indentation studies, we have shown that pores were considered to be appropriate for nutrient supply without compromising the strength of sample while in contact with physiological fluid. After SBF immersion test, porous surface was found to be useful for nucleation of apatite crystals, hence increasing the feasibility and bioactivity of sample. However, our quasi-dynamic study showed less crystallization but had significant formation of apatite layer. Overall, the in vitro analyses show that HAp coated e-glass leads to significant improvement of implant properties in terms of biocompatibility, cell viability and proliferation, osteoinductivity and osteoconductivity. HAp coating of e-glass can potentially be utilized in fabricating durable and strong bioactive non-metallic implants and tissue engineering scaffolds.

Effect of substrate topography on the regulation of human corneal stromal cells.

Optimal functionality of native corneal stroma depends on a well-ordered arrangement of extracellular matrix (ECM). To develop an in vitro corneal model, replication of the corneal in vivo microenvironment is needed. In this study, the impact of topographic cues on keratocyte phenotype is reported. Photolithography and polymer moulding were used to fabricate microgrooves on polydimethylsiloxane (PDMS) 2-2.5 µm deep and 5 µm, 10 µm, or 20 µm in width. Microgrooves constrained the cells body, compressed nuclei and led to cytoskeletal reorganization. It also influenced the concentration of actin filaments, condensation of chromatin and cell proliferation. Cells became more spread and actin filament concentration decreased as the microgroove width increased. Relationships were also demonstrated between microgroove width and cellular processes such as adhesion, migration and gene expression. Immunocytochemistry and gene expression (RT-PCR) analysis showed that microgroove width upregulated keratocyte specific genes. A microgroove with 5 µm width led to a pronounced alignment of cells along the edges of the microchannels and better supported cell polarization and migration compared with other microgroove widths or planar substrates. These findings provide important fundamental knowledge that could serve as a basis for better-controlled tissue growth and cell-engineering applications for corneal stroma regeneration through topographical patterns.

Potential for combined delivery of riboflavin and all-trans retinoic acid, from silk fibroin for corneal bioengineering.

Millions of people worldwide suffer from vision impairing conditions resulting from corneal injury or disease. Silk fibroin (SF) is an emerging biopolymer that has been used for several applications including the fabrication of bioengineered corneas and ocular prostheses. To improve the cell response to SF, riboflavin (RF) and all-trans retinoic acid (RA) were coupled onto SF matrices. RF is a photo-initiator that has previously been combined with ultraviolet light to crosslink corneal collagen while RA has been used to regulate the phenotype of corneal stromal cells and their extracellular matrix deposition. Different concentrations of RF and RA were respectively photo-crosslinked and covalently bound through carbodiimide coupling onto 2% SF matrices. The effect of incorporating these molecules on the physical, chemical and mechanical properties of the matrices was evaluated. The biological response of human corneal stromal cells to the matrices was examined using cellular adhesion assays, proliferation assays, cytoskeleton staining, gene expression analysis and immunocytochemical staining. RF and RA both led to changes in the surface nanostructure and hydrophilicity while just RF increased the material stiffness. Cells cultured on the matrices containing both biomolecules displayed improved cellular proliferation, increased GAG deposition and increased expression of keratocyte genes that are normally associated with healthy corneal stromal tissue. These in vitro studies serve as a starting point for the optimization of loading bioactive molecules on SF based matrices for formulating clinically relevant ocular implants.

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.

Effect of different mineralization processes on in vitro and in vivo bone regeneration and osteoblast-macrophage cross-talk in co-culture system using dual growth factor mediated non-mulberry silk fibroin grafted poly (Є-caprolactone) nanofibrous scaffold.

This study evaluates mineralized nanofibrous polymeric scaffolds at supporting in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and in vivo bone tissue regeneration. Co-electrospin, alternative soaking, and electrodeposition were used to introduce hydroxyapatite in non-mulberry silk fibroin grafted poly(Є-caprolactone) nanofibrous scaffolds. Bone morphogenic protein-2 and Transforming growth factor-ß, at a potency ratio of 1:1, are covalently coupled onto the scaffolds. hMSCs proliferation and interactions are studied through MTT and Alamar blue assay and scanning electron and confocal microscopy. Alkaline phosphatase activity, mineralization assays, and real-time PCR studies substantiate hMSCs' osteogenic differentiation. Co-cultures of human macrophages and osteoblasts exhibit insignificant pro-inflammatory cytokines production. In vivo trials are conducted in rabbit femur (distal metaphysis region). Bone regeneration ability of the scaffolds' is assessed using chronological radiography, micro-CT analysis, host tissue immuno-compatibility, histology, scanning electron microscope imagery, and fluorochrome labelling. In vitro and in vivo characterizations for osteogenesis and osseointegration show best results for scaffolds mineralized by electrodeposition, followed by alternate soaking and co-electrospinning. Non-mulberry silk fibroin grafted poly(Є-caprolactone) nanofibrous scaffold, mineralized by electrodeposition, could provide promising platform for bone healing and regeneration.

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.

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.

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.

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.

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 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.