In Situ Synthesis of Keratin and Melanin Chromophoric Submicron Particles.

In humans, melanin plays an esthetic role, dictating hair and skin color and traits, while keratin is the protein that comprises most of the epidermis layer. Eumelanin and pheomelanin are types of melanin synthesized from the same building blocks via enzymatic oxidation. Pheomelanin has an additional building block, cysteine amino acid, which affects its final structure. Keratin contains high cysteine content, and by exploiting free thiols in hydrolyzed keratin, we demonstrate the formation of keratin-melanin (KerMel) chromophoric submicron particles. Cryo-TEM analyses found KerMel particle sizes to be 100-300 nm and arranged in the form of a central keratin particle with polymerized l-dopa chains. Attenuated total reflection (ATR)-FTIR, UV-vis, and fluorescence measurements identified new chemical bonds, indicating the formation of KerMel particles. Finally, KerMel replicated natural skin tones and proved cytocompatibility for human epidermal keratinocytes at concentrations below 0.1 mg/mL. Taken together, KerMel is a novel, tunable material that has the potential to integrate into the cosmetic industry.

Biocompatible, Resilient, and Tough Nanocellulose Tunable Hydrogels.

Hydrogels have been proposed as potential candidates for many different applications. However, many hydrogels exhibit poor mechanical properties, which limit their applications. Recently, various cellulose-derived nanomaterials have emerged as attractive candidates for nanocomposite-reinforcing agents due to their biocompatibility, abundance, and ease of chemical modification. Due to abundant hydroxyl groups throughout the cellulose chain, the grafting of acryl monomers onto the cellulose backbone by employing oxidizers such as cerium(IV) ammonium nitrate ([NH4]2[Ce(NO3)6], CAN) has proven a versatile and effective method. Moreover, acrylic monomers such as acrylamide (AM) may also polymerize by radical methods. In this work, cerium-initiated graft polymerization was applied to cellulose-derived nanomaterials, namely cellulose nanocrystals (CNC) and cellulose nanofibrils (CNF), in a polyacrylamide (PAAM) matrix to fabricate hydrogels that display high resilience (~92%), high tensile strength (~0.5 MPa), and toughness (~1.9 MJ/m3). We propose that by introducing mixtures of differing ratios of CNC and CNF, the composite's physical behavior can be fine-tuned across a wide range of mechanical and rheological properties. Moreover, the samples proved to be biocompatible when seeded with green fluorescent protein (GFP)-transfected mouse fibroblasts (3T3s), showing a significant increase in cell viability and proliferation compared to samples comprised of acrylamide alone.

Biomaterial Design Inspired by Regenerative Research Organisms.

The efficacy of implanted biomaterials is largely dependent on the response of the host's immune and stromal cells. Severe foreign body response (FBR) can impede the integration of the implant into the host tissue and compromise the intended mechanical and biochemical function. Many features of FBR, including late-stage fibrotic encapsulation of implants, parallel the formation of fibrotic scar tissue after tissue injury. Regenerative organisms like zebrafish and salamanders can avoid fibrosis after injury entirely, but FBR in these research organisms is rarely investigated because their immune competence is much lower than humans. The recent characterization of a regenerative mammal, the spiny mouse (Acomys), has inspired us to take a closer look at cellular regulation in regenerative organisms across the animal kingdom for insights into avoiding FBR in humans. Here, we highlight how major features of regeneration, such as blastema formation, macrophage polarization, and matrix composition, can be modulated across a range of regenerative research organisms to elucidate common features that may be harnessed to minimize FBR. Leveraging a deeper understanding of regenerative biology for biomaterial design may help to reduce FBR and improve device integration and performance.

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.

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.

Silk fibroin for skin injury repair: Where do things stand?

Several synthetic and natural materials are used in soft tissue engineering and regenerative medicine with varying degrees of success. Among them, silkworm silk protein fibroin, a naturally occurring protein-based biomaterial, exhibits many promising characteristics such as biocompatibility, controllable biodegradability, tunable mechanical properties, aqueous preparation, minimal inflammation in host tissue, low cost and ease of use. Silk fibroin is often used alone or in combination with other materials in various formats and is also a promising delivery system for bioactive compounds as part of such repair scenarios. These properties make silk fibroin an excellent biomaterial for skin tissue engineering and repair applications. This review focuses on the promising characteristics and recent advances in the use of silk fibroin for skin wound healing and/or soft-tissue repair applications. The benefits and limitations of silk fibroin as a scaffolding biomaterial in this context are also discussed. STATEMENT OF SIGNIFICANCE: Silk protein fibroin is a natural biomaterial with important biological and mechanical properties for soft tissue engineering applications. Silk fibroin is obtained from silkworms and can be purified using alkali or enzyme based degumming (removal of glue protein sericin) procedures. Fibroin is used alone or in combination with other materials in different scaffold forms, such as nanofibrous mats, hydrogels, sponges or films tailored for specific applications. The investigations carried out using silk fibroin or its blends in skin tissue engineering have increased dramatically in recent years due to the advantages of this unique biomaterial. This review focuses on the promising characteristics of silk fibroin for skin wound healing and/or soft-tissue repair applications.

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.

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.

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.

Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture.

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Exploration of the tight structural-mechanical relationship in mulberry and non-mulberry silkworm silks.

The Bombyx mori silkworm is well known as it has been bred by our ancestors with mulberry tree leaves for thousands of years. However, Bombyx mori is not the only silkworm that can produce silk, many other kinds of silkworms can also make silks for commercial use. In this research, we compare the mechanical properties of five different commercial silk fibres including domesticated mulberry Bombyx mori, non-mulberry semi-domesticated eri Samia ricini, and wild tropical tasar Antheraea mylitta and muga Antheraea assamensis. The results demonstrate that the non-mulberry silk fibres have a relatively high extensibility as compared to the mulberry silk fibres. In the meantime, the non-mulberry silk fibres show comparatively unique toughness to the mulberry silk fibres. Synchrotron radiation FTIR microspectroscopy, synchrotron radiation wide angle X-ray diffraction, and Raman dichroism spectroscopy are used to analyze the structural differences among the five species of silk fibres comprehensively. The results clearly show that the mechanical properties of both mulberry and non-mulberry silk fibres are closely related to their structures, such as ß-sheet content, crystallinity, and the molecular orientation along the fibre axis. This study aims to understand the differences in the structural and mechanical properties of different mulberry and non-mulberry silk fibres, which are of importance to the related research on understanding and utilizing the non-mulberry silk as a biomaterial. We believe these investigations not only provide insight into the biology of silk fibroins from the non-mulberry silkworms but also offer guidelines for further biomimetic investigations into the design and manufacture of artificial silk protein fibres with novel morphologies and associated material properties for future use in different fields like bioelectronics, biomaterials and biomedical devices.