Banani Kundu

Silk fibroin biomaterials for tissue regenerations

Regeneration of tissues using cells, scaffolds and appropriate growth factors is a key approach in the treatments of tissue or organ failure. Silk protein fibroin can be effectively used as a scaffolding material in these treatments. Silk fibers are obtained from diverse sources such as spiders, silkworms, scorpions, mites and flies. Among them, silk of silkworms is a good source for the development of biomedical device. It possesses good biocompatibility, suitable mechanical properties and is produced in bulk in the textile sector. The unique combination of elasticity and strength along with mammalian cell compatibility makes silk fibroin an attractive material for tissue engineering. The present article discusses the processing of silk fibroin into different forms of biomaterials followed by their uses in regeneration of different tissues. Applications of silk for engineering of bone, vascular, neural, skin, cartilage, ligaments, tendons, cardiac, ocular, and bladder tissues are discussed. The advantages and limitations of silk systems as scaffolding materials in the context of biocompatibility, biodegradability and tissue specific requirements are also critically reviewed.

Silk protein fibroin from Antheraea mylitta for cardiac tissue engineering

The human heart cannot regenerate after an injury. Lost cardiomyocytes are replaced by scar tissue resulting in reduced cardiac function causing high morbidity and mortality. One possible solution to this problem is cardiac tissue engineering. Here, we have investigated the suitability of non-mulberry silk protein fibroin from Indian tropical tasar Antheraea mylitta as a scaffold for engineering a cardiac patch in vitro. We have tested cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication and contractility of 3-days postnatal rat cardiomyocytes on silk fibroin. Our data demonstrate that A. mylitta silk fibroin exhibits similar properties as fibronectin, a component of the natural matrix for cardiomyocytes. Comparison to mulberry Bombyx mori silk protein fibroin shows that A. mylitta silk fibroin is superior probably due to its RGD domains. 3D scaffolds can efficiently be loaded with cardiomyocytes resulting in contractile patches. In conclusion, our findings demonstrate that A. mylitta silk fibroin 3D scaffolds are suitable for the engineering of cardiac patches.

Nonmulberry silk biopolymers

The silk produced by silkworms are biopolymers and can be classified into two types--mulberry and nonmulberry. Mulberry silk of silkworm Bombyx mori has been extensively explored and used for century old textiles and sutures. But for the last few decades it is being extensively exploited for biomedical applications. However, the transformation of nonmulberry silk from being a textile commodity to biomaterials is relatively new. Within a very short period of time, the combination of load bearing capability and tensile strength of nonmulberry silk has been equally envisioned for bone, cartilage, adipose, and other tissue regeneration. Adding to its advantage is its diverse morphology, including macro to nano architectures with controllable degradation and biocompatibility yields novel natural material systems in vitro. Its follow on applications involve sustained release of model compounds and anticancer drugs. Its 3D cancer models provide compatible microenvironment systems for better understanding of the cancer progression mechanism and screening of anticancer compounds. Diversely designed nonmulberry matrices thus provide an array of new cutting age technologies, which is unattainable with the current synthetic materials that lack biodegradability and biocompatibility. Scientific exploration of nonmulberry silk in tissue engineering, regenerative medicine, and biotechnological applications promises advancement of sericulture industries in India and China, largest nonmulberry silk producers of the world. This review discusses the prospective biomedical applications of nonmulberry silk proteins as natural biomaterials.

Silk sericin/polyacrylamide in situ forming hydrogels for dermal reconstruction

In situ forming tissue sealants are advantageous due to ease in application, complete coverage of defect site and assured comfort levels to patients. The interconnected three-dimensional hydrophilic networks perfectly manage typical dermal wounds by suitably scaffolding skin fibroblast, diffusing the nutrients, therapeutics and exudates while still maintaining an adequately moist environment. We evaluate the cell homing ability of semi-interpenetrating non-mulberry tropical tasar silk sericin/polyacrylamide hydrophilic network with a keen understanding of its network characteristics and correlation of protein concentration with the performance as cell scaffold. Interconnectivity of porous networks observed through scanning electron micrograph revealed pore sizes ranging from 23 to 52 μm. The enhanced β-sheet content with the increasing sericin concentration in far red spectroscopy study supported their corresponding improved compressive strength. These semi-interpenetrating networks were found to possess a maximum fluid uptake of 112% of its weight, hence preventing the accumulation of exudates at the wound area. The present systems appear to possess characteristics like rapid gelation (~5min) at 37 °C, 98% porosity enabling the migration of fibroblasts during healing (observed through confocal and scanning electron micrographs), cell adhesion together with the absence of any cyto-toxic effect suggesting its potential as in situ tissue sealants. The compressive strength up to 61 kPa ensured ease in handling even when wet. The results prove the suitability to use non-mulberry tasar cocoon silk sericin/polyacrylamide semi-interpenetrating network as a reconstructive dermal sealant.

Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms

The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.

Isolation and processing of silk proteins for biomedical applications

Silk proteins of silkworms are chiefly composed of core fibroin protein and glycoprotein sericin that glues fibroin. Unique mechanical properties, cyto-compatibility and controllable biodegradability facilitate the use of fibroin in biomedical applications. Sericin serves as additive in cosmetic and food industries, as mitotic factor in cell culture media, anti-cancerous drug, anticoagulant and as biocompatible coating. For all these uses; aqueous solutions of silk proteins are preferred. Therefore, an accurate understanding of extraction procedure of silk proteins from their sources is critical. A number of protocols exist, amongst which it is required to settle a precise and easy one with desired yield and least down-stream processing. Here, we report extraction of proteins employing methods mentioned in literature using cocoons of mulberry and nonmulberry silks. This study reveals sodium carbonate salt-boiling system is the most efficient sericin extraction procedure for all silk variants. Lithium bromide is observed as the effective fibroin dissolution system for mulberry silk cocoons; whereas heterogeneous species-dependent result is obtained in case of nonmulberry species. We further show the effect of common post processing on nanoscale morphology of mulberry silk fibroin films. This knowledge eases the adoption and fabrication of silk biomaterials in devices and therapeutic delivery systems.

Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering.

To fabricate hard tissue implants with bone-like structure using a biomimetic mineralization method is drawing much more attentions in bone tissue engineering. The present work focuses in designing 3D silk fibroin hydrogel to modulate the nucleation and growth of hydroxyapatite crystals via a simple ion diffusion method. The study indicates that Ca(2+) incorporation within the hydrogel provides the nucleation sites for hydroxyapatite crystals and subsequently regulates their oriented growth. The mineralization process is regulated in a Ca(2+) concentration- and minerlization time-dependent way. Further, the compressive strength of the mineralized hydrogels is directly proportional with the mineral content in hydrogel. The orchestrated organic/inorganic composite supports well the viability and proliferation of human osteoblast cells; improved cyto-compatibility with increased mineral content. Together, the present investigation reports a simple and biomimetic process to fabricate 3D bone-like biomaterial with desired efficacy to repair bone defects.

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.

Ion-induced fabrication of silk fibroin nanoparticles from Chinese oak tasar Antheraea pernyi

Silk protein fibroin in nanoparticles form is a promising material for drug delivery due to its pleiotropic properties, including biocompatibility, biodegradability, ease in fabrication into smaller diameters, high bioavailability, and therapeutic retention at target sites. In the present study, silk nanoparticles are fabricated from regenerated fibroin solution of the Chinese temperate oak tasar Antheraea pernyi by novel ion-induced self-assembly in a very short time under mild conditions. The resultant fibroin nanoparticles range in size from 100 to 500 nm. The molecular conformation of regenerated fibroin changes from α-helical to a β-sheet structure as a rapid function of the ionic strength and the hydrophobic and electrostatic interactions. The mild conditions are potentially advantageous for the encapsulation of sensitive drugs and therapeutic molecules such as doxorubicin hydrochloride, an amphiphilic anticancer therapeutic. In vitro release of doxorubicin from nanoparticles is pH sensitive, with approx. 65% doxorubicin remaining in the fibroin nanoparticles after 11 days. The activity of fibroin nanoparticles on hepatomas indicates the efficacy of the fibroin nanoparticles to maintain the bioactivity of the loaded doxorubicin and impart a dose-dependent cell growth inhibition. The results suggest that Chinese temperate oak tasar silk fibroin nanoparticles can be used as a sustained drug delivery vehicle.

Biomimetic Designing of Functional Silk Nanotopography Using Self-assembly

In nature inorganic-organic building units create multifunctional hierarchical architectures. Organic silk protein is particularly attractive in this respect because of its micro-nanoscale structural blocks that are attributed to sophisticated hierarchical assembly imparting flexibility and compressibility to designed biohybrid materials. In the present study, aqueous silk fibroin is assembled to form nano/microtopography on inorganic silica surface via a facile diffusion-limited aggregation process. This process is driven by electrostatic interaction and only possible at a specified aminated surface chemistry. The self-assembled topography depends on the age and concentration of protein solution as well as on the surface charge distribution of the template. The self-assembled silk trails closely resemble natural cypress leaf architecture, which is considered a structural analogue of neuronal cortex. This assembled surface significantly enhances anchorage of neuronal cell and cytoskeletal extensions, providing an effective nano/microtopographical cue for cellular recognition and guidance.