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.

Identification of a novel proline-rich antimicrobial protein from the hemolymph of Antheraea mylitta.

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.

Sericin-chitosan-glycosaminoglycans hydrogels incorporated with growth factors for in vitro and in vivo skin repair.

Globally, skin repair costs billion dollars per annum. Diversified matrices are fabricated to address this important area of healthcare. Most common limitations associated with them are the inflated production cost and insufficient functional repair. Our work explores the fabrication and potential utilization of Antheraea mylitta silk protein sericin (possessing inherent anti-bacterial and antioxidant properties) based hydrogels for skin tissue. The integrity of the hydrogels is achieved by combining sericin, chitosan (provide anti-bacterial and structural support), and glycosaminoglycans (component of biologically formed extracellular matrix). The hydrogels are functionalized by incorporation of vascular endothelial growth factor and transforming growth factor-ß. They exhibit enhanced cellular functions in terms of their growth, production of matrix metalloproteinase, and collagen along with the recovery of impairment and the reconstruction of the lost dermal tissue. The in vivo biocompatibility analyses reveal that sericin-containing hydrogels promote the repair of skin tissue, angiogenesis, and illicit minimal immune response. These unique hydrogels mimicking the naturally occurring skin tissue and imparting additional beneficial features provide an appropriate physical environment and biological cues for the promotion of skin tissue repair.

Purification and characterization of a novel antimicrobial peptide (QAK) from the hemolymph of Antheraea mylitta.

Antheraea mylitta, a tropical non-mulberry silkworm, is cultivated for tasar silk production in India. Several defense molecules including few antimicrobial peptides (AMPs) and proteins have been identified from this insect. Here, we have isolated and purified an antimicrobial tri-peptide by sequential chromatographic separation procedures. The amino acid sequence of the peptide was determined as NH2-Gln-Ala-Lys-COOH (QAK) using MALDI MS/MS fragmentation analysis. Further, the peptide was synthesized in vitro following solid phase chemistry of peptide synthesis and acetylated by acetic anhydride reaction. Antimicrobial activities of non-acetylated and acetylated QAK were tested against both Escherichia coli and Staphylococcus aureus bacteria. Acetylated peptide inhibited bacterial growth more effectively and its minimum inhibitory concentration (MICs) was found lower than non-acetylated peptide. SEM studies revealed more membrane damage and release of intracellular materials like ß-galactosidase enzyme from acetylated peptide treated bacteria in comparison to non-acetylated QAK. At MIC, acetylated peptide did not show any significant hemolytic activity against rabbit erythrocytes. The results suggest that acetylated-QAK is a promising new antimicrobial peptide and can be used for therapeutic purpose.

Colistin Induced Assortment of Antimicrobial Resistance in a Clinical Isolate of Acinetobacter baumannii SD01.

BACKGROUND: Colistin was considered as the most effective antibiotic against Acinetobacter baumannii, a widely-known opportunistic pathogen. In recent years, a number of colistin resistant strains have also been reported. OBJECTIVE: This work is commenced to investigate the contribution of efflux pumps toward resistance to colistin-like cyclic polypeptide antibiotics, since the efflux pumps serve as the escape routes leading to drug-resistance. METHODS: RNA was extracted from A. baumannii isolates cultured from samples procured by tracheal aspiration of infected patients. The expressions of gene(s) that played major roles in the regulation of efflux pump families and involvement of integron systems were studied using real time PCR. Antimicrobial susceptibility tests were conducted to investigate antibiotic resistance of the isolates. RESULTS: It was observed that genes coding for sugE, ydhE, ydgE, mdfA, ynfA and tolC significantly contributed to resistance against colistin antibiotics, however, no significant transcriptional change was observed in the efflux pump, MexAB-OprM. Results suggest that A. baumanii readily pumps out colistin via efflux pumps belonging to MATE and SMR family. CONCLUSION: Integral role of efflux pumps and integron 1 genetic system was elucidated towards evolution of multi-drug resistant strain(s). Therefore, for accurate therapeutics, an early detection of efflux genes is crucial before prescribing against colistin resistant A. baumanii.

Coevolution of Resistance Against Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are produced by all forms of life, ranging from eukaryotes to prokaryotes, and they are a crucial component of innate immunity, involved in clearing infection by inhibiting pathogen colonization. In the recent past, AMPs received high attention due to the increase of extensive antibiotic resistance by these pathogens. AMPs exhibit a diverse spectrum of activity against bacteria, fungi, parasites, and various types of cancer. AMPs are active against various bacterial pathogens that cause disease in animals and plants. However, because of the coevolution of host and pathogen interaction, bacteria have developed the mechanisms to sense and exhibit an adaptive response against AMPs. These resistance mechanisms are playing an important role in bacterial virulence within the host. Here, we have discussed the different resistance mechanisms used by gram-positive and gram-negative bacteria to sense and combat AMP actions. Understanding the mechanism of AMP resistance may provide directions toward the development of novel therapeutic strategies to control multidrug-resistant pathogens.

Identification of a novel humoral antifungal defense molecule in the hemolymph of tasar silkworm Antheraea mylitta.

Humoral defenses are the major components of insect innate immune system that include the production of several soluble effector molecules from fat body and hemocytes, and released in to the hemolymph upon microbial infection. Hemolymph was collected from the fungal immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, extracted with a mixture of solvent (methanol/glacial acetic acid/water) and fractionated through RP-HPLC. Several fractions were collected, lyophilized and their antifungal activity was tested against Candida albicans. Only the fraction showing strong antifungal activity was further purified via gel filtration chromatography and the purity of active compound was confirmed by thin layer chromatography which showed only single spot after staining with ninhydrin. The molecular mass of this purified compound was determined by high resolution mass spectrometry as 531 Da and analysis of 1H and 13C NMR spectral data along with mass fragmentation pattern indicated the probable structure of the isolated compound as symmetric bis-decanoate derivative. Scanning electron microscopic study revealed that the compound degraded fungal cell wall leading to its lysis and may be the major target for its antifungal activity. These results indicate that presence of this compound in the hemolymph of A. mylitta provides defense against fungal infection.

Nonmulberry silk protein sericin blend hydrogels for skin tissue regeneration - in vitro and in vivo.

The damage to the skin is most prominent and evident as it is our first line of defense and unremittingly under attack by biological and environmental factors. The restoration of the skin is dependent on the extent of the injury. To explore the prospects of new biomimetic material, bi-layered skin construct is fabricated in vitro with nonmulberry silk protein sericin and chitosan hydrogels using human dermal fibroblasts and keratinocytes. The in vitro analysis of the hydrogels showed enhanced adhesion, proliferation, and migration of skin cells with coordinated interaction amongst themselves leading to the synthesis of collagen IV and matrix metalloproteinase (MMP2 and MMP9). The in vivo evaluation indicates the regeneration of skin with densely packed collagen and formation of matured blood vessels in the animals implanted with sericin containing hydrogels. Moreover, the local and systemic immune response determined in vivo exhibits the biosafety of sericin based hydrogels. In addition, the cross-sectional analysis of the implanted hydrogels displays infiltration of the skin tissue cells into the hydrogels marking their biocompatibility and non-toxicity. The cumulative analysis of the in vitro and in vivo observations demonstrates that the sericin based hydrogels are non-inflammatory, supports the regeneration and repair of the skin tissue.

Curd-Peptide Based Novel Hydrogel Inhibits Biofilm Formation, Quorum Sensing, Swimming Mortility of Multi-Antibiotic Resistant Clinical Isolates and Accelerates Wound Healing Activity.

The search for a bioactive natural antibacterial agent with wound healing properties is a common practice for the development of new-generation molecules. Antimicrobial peptides are a good alternative to antibiotics and easy-to-form hydrogels under self-assembled conditions without pH adjustment. With this in mind, the peptide pool was extracted from a formulated curd composed of a blend of probiotic bacteria such as Streptococcus thermophilus, Lactobacillus casei, and Bifidobacterium bifidum at an optimized ratio of 7:1:2. The water content of curd was collected by the drainage column, centrifuged, filtered through a 0.45-µM filter, and used for hydrogel preparation. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis confirmed the presence of peptide pool in the extracted water. The prepared hydrogel was freeze dried, and its effect on biofilm formation, swarming mortality, antimicrobials, wound healing, and biocompatibility was subsequently verified. Transmission electron microscope (TEM) and scanning electron microscope (SEM) images revealed the fibrous network of peptides after self-assembly with non-polar n-hexane solvent and a porous structure after drying, respectively. The observed biocompatibility, antimicrobial activity, and strong wound healing activity of the developed curd-based hydrogel have opened a new platform for antibacterial ointment formulation.

Investigation of diversity and dominance of fungal biota in stored wheat grains from governmental warehouses in West Bengal, India.

BACKGROUND: Fungal infestation is a leading cause of qualitative and quantitative deterioration of stored wheat grains. Limited information is available on the spatial distribution of fungal biota associated with stored wheat grains in India. Fungi were isolated and characterized from nine stored wheat grain samples in three warehouses of the Food Corporation of India, located in three agro-climatic zones (Paschim Medinipur, Bankura and Purulia) of West Bengal in India. RESULTS: Maximum density and fungal diversity were observed in dichloran glycerol agar (DG-18) medium and the number increased with the increase of storage duration. Samples collected from Purulia showed maximum fungal diversity than that from Bankura and Paschim Medinipur. A total of 284 fungal isolates were obtained, classified into 29 operational taxonomic units (based on amplified ribosomal DNA restriction analysis of 18S and internal transcribed spacer sequences), and identified as 24 different fungal species. The majority of fungal isolates belonged to Aspergillus flavus (35%) followed by Rhizopus oryzae (13%) and Eurotium amstelodami (9%). Aspergillopepsin O (PEPO) gene and aflatoxin biosynthetic pathway gene, nor-1, were amplified by polymerase chain reaction (PCR) from 91% and 71% of Aspergillus flavus isolates, respectively, indicating their aflatoxin producing ability. Aflatoxin production was further confirmed by ammonia vapour test, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). CONCLUSION: The presence of toxigenic fungi in stored wheat grain emphasizes the necessity of quarantine measures of stored grains before placing them in the public domain to save consumers from health hazards. © 2019 Society of Chemical Industry.

Prospects of nonmulberry silk protein sericin-based nanofibrous matrices for wound healing - In vitro and in vivo investigations.

Recently, the progress in biomaterials for biomedical applications brings the focus of the research community toward nanomaterials. The nanofibrous matrices offer certain advantages (structural similarity to extracellular matrix, high surface area-to-volume ratio, increased elasticity, biostability, and strength) compared to other prevalent type of materials. This affirms their superiority and flexibility to be used in regenerative medicine. We have fabricated nonmulberry (Antheraea mylitta) silk protein sericin-based nanofibrous matrices (fiber thickness; 80-400 nm) with improved mechanical strength and desired stability (>4 weeks) as required for tissue reconstruction. These matrices support the adhesion, proliferation, and cellular interconnection of human keratinocytes. These are minimally hemolytic, nonimmunogenic, and capable of wound healing in vivo. Antibiotic (cephalexin hydrate [CH])-loaded nanofibrous matrices accelerate the full-thickness wound repair with minimal inflammation and without any signs of infection. The histological analysis authenticates skin restoration with re-epithelialization, generation of associated skin appendages, and synthesis of dense collagen fibrils. In addition, analysis of inflammatory genes and immunohistochemical assays have proved their biocompatibility and wound healing potential. Angiogenesis is also prevalent in the animal tissue treated with nanofibrous matrices. The results of in vitro and in vivo experimentations indicate a clear prospect of the fabricated sericin-based nanofibrous matrices to be used for skin regeneration. STATEMENT OF SIGNIFICANCE: Nonmulberry silk protein sericin-based nanofibrous matrix is a useful biomaterial for wound healing, collagen production, and skin tissue repair. It has been used in different formulations including hydrogels and nanofibrous membranes with chitosan (CS) and polyvinyl alcohol (PVA). No experiments have been carried out to evaluate sericin-based nanofibrous membranes for skin tissue engineering application. The present study shows that the nanofibrous matrices fabricated by electrospinning nonmulberry silk protein sericin with CS and PVA mimic the architectural environment of the extracellular matrix fibrils. These matrices are minimally hemolytic, are nonimmunogenic, and support better growth of human keratinocytes in vitro and wound healing in vivo with re-epithelialization of the skin tissue and angiogenesis. This work indicates that these nonmulberry sericin-based nanofibrous matrices with CS may be used as an ideal physical environment and biological cues for the promotion of skin tissue reconstruction and repair.

Transcriptional regulation of human defense peptides: a new direction in infection control.

While antibiotics remain as a major therapy against life threatening pathogenic infections, they often lead to side effects like rashes, gastrointestinal and central nervous system reactions to serious allergies or organ damage. These adverse effects alongside the emergence of multi-antibiotic resistant bacteria and the decline in the development of new antibiotics, have posed a serious impediment for effective antibiotic therapy. A paradigm shift in attitudes has led us to think about the possibility of controlling infections with the indigenous antimicrobial peptides synthesized by human beings. It has been observed that few transcription factors can stimulate more than three dozen defense peptides in the human system. Hence, during the infection stage, if we can induce these common factors, most of the infections could be healed from inside without the administration of any antibiotics. The efficiency of such peptides is being proven in clinical tests leading to the development of drugs.

"Omics" in oral cancer: New approaches for biomarker discovery.

OBJECTIVES: In this review paper, we explored the application of "omics" approaches in the study of oral cancer (OC). It will provide a better understanding of how "omics" approaches may lead to novel biomarker molecules or molecular signatures with potential value in clinical practice. A future direction of "omics"-driven research in OC is also discussed. METHODS: Studies on "omics"-based approaches [genomics/proteomics/transcriptomics/metabolomics] were investigated for differentiating oral squamous cell carcinoma,oral sub-mucous fibrosis, oral leukoplakia, oral lichen planus, oral erythroplakia from normal cases. Electronic databases viz., PubMed, Springer, and Google Scholar were searched. RESULTS: One eighty-one studies were included in this review. The review shows that the fields of genomics, transcriptomics, proteomics, and metabolomics-based marker identification have implemented advanced tools to screen early changes in DNA, RNA, protein, and metabolite expression in OC population. CONCLUSIONS: It may be concluded that despite advances in OC therapy, symptomatic presentation occurs at an advanced stage, where various curative treatment options become very limited. A molecular level study is essential for detecting an OC biomarker at an early stage. Modern "Omics" strategies can potentially make a major contribution to meet this need.

Chloramphenicol-borate/boronate complex for controlling infections by chloramphenicol-resistant bacteria.

Increasing bacterial resistance to antibiotics is a pressing problem worldwide, with many health organisations prioritizing this issue. Whilst there is a desperate need for new effective antimicrobials, it is also important to understand the mechanisms and epidemiology of the resistant pathogens currently present in the community. Chloramphenicol is one such well known antibiotic which had lost its efficacy due to bacterial resistance. In this paper, we report the design, synthesis, and bio-studies of novel chloramphenicol-borate/boronate derivatives which showed the ability to control the infections caused by chloramphenicol-resistant bacteria. Activity profiling against P. aeruginosa strain EXR1 with catB gene indicated the inability of acetyl transferase to acetylate the chloramphenicol-borate/boronate complex, unlike chloramphenicol. Results obtained from the antimicrobial assays were further rationalized by molecular docking studies. The latter revealed that the probable reason for the enhanced antibacterial activity may be attributed to the change in the binding site of chloramphenicol-borate/boronate with chloramphenicol acetyl transferase (CAT) with respect to chloramphenicol itself. Hemolytic and genotoxic studies established the reduced toxicity of these synthetic derivatives with respect to chloramphenicol.

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.

Non-immunogenic, porous and antibacterial chitosan and Antheraea mylitta silk sericin hydrogels as potential dermal substitute.

Limitation of existing grafts including restricted donor site, risks of immune reactions, infectious diseases and high cost alarms the growing need of natural, cost effective and functional graft as the dermal substitute. We fabricate stable (>6 weeks) and porous (57.23-75.22µm) yet flexible (in variable pH) matrices using Antheraea mylitta sericin crosslinked with well known biocompatible polysaccharide chitosan by natural crosslinker (genipin) without using any harsh chemical. The fabricated matrices are characterized in terms of chemical modifications (Fourier transform infrared spectroscopy), crystallinity (X-ray diffraction), swelling, degradability and thermal stability. The hydrogels show good adhesion, migration, proliferation and viability of human dermal fibroblasts. The matrices cause no significant immune response of inflammatory cytokines (TNF-α and IL-1ß) and hemolysis of human blood. These also retain their intrinsic antioxidant (196.1±17.7µM Fe (II)/mg) and antibacterial (8-15mm zone of inhibition) properties. These results indicate their potential as a cost effective and antibacterial dermal substitute.

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.

Elucidation of structural and functional integration of a novel antimicrobial peptide from Antheraea mylitta.

We report here the amino acid sequence of an antimicrobial peptide of Antheraea mylitta (peptide fraction II) effectively killed urinary tract associated MDR E. coli (Dutta et al., 2016), as Gly-Gly-Gly-Gly-Gly-Gly-His-Leu-Val-Ala. The physicochemical and biological properties of this peptide were evaluated by computational analysis and its isoelectric point, grand average of hydropathicity and Boman index values were found to be 6.74, 0.42 and -1.17kcal/mol, respectively. One valid model of peptide fraction II was constructed, that contains two antiparallel ß sheets with a hairpin and appeared as 'U' shaped structure. The glycine rich composition (Gly1, Gly5, Gly6 and Ala10) facilitates mostly for its flexibility or dynamicity, and in its other wing, aggregation prone residues (Leu8, Val9, Ala10) triggered its auto-aggregations when contacted only with the microbial membrane. We employed simulation of peptide binding on the membrane, showed stable and deep insertion of peptide fraction II into the membrane through its hydrophobic tail (up to 3.3±1.46Å). Molecular docking study with Patchdock server revealed that this peptide could interact with the lipid aliphatic chain of 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE) bilayer and may linked to membrane distortion as we have reported earlier. Further, the studied peptide has been predicted not to exhibit any antigenicity and non-responsive to RBC membrane. These data for the first time provide new insights of an antimicrobial peptide from silkworm A. mylitta and it may serve as the template for the design of novel peptide antibiotics from this group of insect against MDR Gram-negative bacteria.

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.