Development of bilayered porous silk scaffolds for thymus bioengineering.

The thymus coordinates the development and selection of T cells. It is structured into two main compartments: the cortex and the medulla. The replication of such complex 3D environment has been challenged by bioengineering approaches. Nevertheless, the effect of the scaffold microstructure on thymic epithelial cell (TEC) cultures has not been deeply investigated. Here, we developed bilayered porous silk fibroin scaffolds and tested their effect on TEC co-cultures. The small and large pore scaffolds presented a mean pore size of 84.33 ± 21.51 µm and 194.90 ± 61.38 µm, respectively. The highly porous bilayered scaffolds presented a high water absorption and water content (> 94 %), together with mechanical properties in the range of the native tissue. TEC (i.e., medullary (mTEC) and cortical (cTEC) cell lines) proliferation is increased in scaffolds with larger pores. The co-culture of both TEC lines in the bilayered porous silk scaffolds presents enhanced cell proliferation and metabolic activity when compared with mTEC in single culture. Also, when the co-culture occurred with cTEC in the small pores layer and mTEC in the large pores layer, a 9.2- and 18.9-fold increase in Foxn1 and Icam1 gene expression in cTEC is evident. These results suggest that scaffold microstructure and the co-culture influence TEC's behaviour. Bilayered silk scaffolds with adjusted microstructure are a valid alternative for TEC culture, having possible applications in advanced thymus bioengineering strategies.

Biomimetic Antibacterial Pro-Osteogenic Cu-Sericin MOFs for Osteomyelitis Treatment.

Osteomyelitis is an inflammation of the bone caused by bacterial infection. It usually develops from broken bones, decayed teeth, or heavily punctured wounds. Multi-drug-resistant bacteria are the major hurdle in the treatment of osteomyelitis. The ever-rising antibiotic resistance even leads to amputations or fatalities as a consequence of chronic osteomyelitis. Hence, a single agent with antibacterial activity as well as bone regenerative properties can serve as a potential off-the-shelf product in the treatment of osteomyelitis. Herein, the antibacterial and pro-osteogenic characteristics of copper sericin (Cu-SER) metal-organic frameworks (MOFs) are reported. Sericin, a silk protein with antibacterial activity and an osteoinduction property, acts as an organic template for the deposition of Cu-SER MOFs, similar to collagen during biomineralization in bone. The MOFs exhibit cytocompatibility and osteogenic activity in a dose-dependent manner, as revealed by cell proliferation (alamarBlue) and mineralization (Alizarin Red S and Energy Dispersive X-ray analysis). The bactericidal activity of Cu-SER MOFs was investigated by scanning electron microscopy and a growth kinetic analysis. Together, the report illuminates the unique phenomenon of Cu-SER MOFs that kill bacteria upon contact while being well-tolerated by primary human cells. Hence, Cu-SER MOFs hold the potential to minimize antibiotic dependence.

In Vitro Cancer Models: A Closer Look at Limitations on Translation.

In vitro cancer models are envisioned as high-throughput screening platforms for potential new therapeutic discovery and/or validation. They also serve as tools to achieve personalized treatment strategies or real-time monitoring of disease propagation, providing effective treatments to patients. To battle the fatality of metastatic cancers, the development and commercialization of predictive and robust preclinical in vitro cancer models are of urgent need. In the past decades, the translation of cancer research from 2D to 3D platforms and the development of diverse in vitro cancer models have been well elaborated in an enormous number of reviews. However, the meagre clinical success rate of cancer therapeutics urges the critical introspection of currently available preclinical platforms, including patents, to hasten the development of precision medicine and commercialization of in vitro cancer models. Hence, the present article critically reflects the difficulty of translating cancer therapeutics from discovery to adoption and commercialization in the light of in vitro cancer models as predictive tools. The state of the art of in vitro cancer models is discussed first, followed by identifying the limitations of bench-to-bedside transition. This review tries to establish compatibility between the current findings and obstacles and indicates future directions to accelerate the market penetration, considering the niche market.

adipoSIGHT in Therapeutic Response: Consequences in Osteosarcoma Treatment.

Chemotherapeutic resistance is a major problem in effective cancer treatment. Cancer cells engage various cells or mechanisms to resist anti-cancer therapeutics, which results in metastasis and the recurrence of disease. Considering the cellular heterogeneity of cancer stroma, the involvement of stem cells is reported to affect the proliferation and metastasis of osteosarcoma. Hence, the duo (osteosarcoma: Saos 2 and human adipose-derived stem cells: ASCs) is co-cultured in present study to investigate the therapeutic response using a nonadherent, concave surface. Staining with a cell tracker allows real-time microscopic monitoring of the cell arrangement within the sphere. Cell-cell interaction is investigated by means of E-cadherin expression. Comparatively high expression of E-cadherin and compact organization is observed in heterotypic tumorspheres (Saos 2-ASCs) compared to homotypic ones (ASCs), limiting the infiltration of chemotherapeutic compound doxorubicin into the heterotypic tumorsphere, which in turn protects cells from the toxic effect of the chemotherapeutic. In addition, genes known to be associated with drug resistance, such as SOX2, OCT4, and CD44 are overexpressed in heterotypic tumorspheres post-chemotherapy, indicating that the duo collectively repels the effect of doxorubicin. The interaction between ASCs and Saos 2 in the present study points toward the growing oncological risk of using ASC-based regenerative therapy in cancer patients and warrants further investigation.

Silk fibroin promotes mineralization of gellan gum hydrogels.

Mineralization is a natural process leading to the formation of mineralized tissue such as bone. The chief mineral component of bone is hydroxyapatite (HAp), which is deposited using an organic template like fibrillar Collagen I under physiological condition. Fibrous silk fibroin is structurally homologous to collagen and acts as nucleation site for HAp mineralization when immersed in simulated body fluid (SBF) or fetal bovine serum (FBS), therefore, considered as popular bone regeneration biomaterial. Hence, the mineralization behavior of silk fibroin self-assembled gellan gum enriched 3D hydrogels is investigated under conditions closer to physiological ones using SBF as well as FBS, and also in presence of cells (e.g. human adipose tissue-derived stem cells, ASCs). Incorporation of silk fibroin induces the mineralization in acellular spongy-like hydrogels in composition dependent manner, confirmed by SEM-EDS analysis. In contrast, ASCs mediated mineralization is found in all hydrogel compositions of 3 weeks post-culture under osteogenic conditions as demonstrated by gene expression profile and Alizarin Red S staining. This is perhaps due to the co-existence of fibroin and FBS together induce cell-mediated mineralization. The blending of fibroin offers cheap alternative strategy to improve or guide the repair of mineralized tissue using gellan gum-based biomaterials.

Curcumin ameliorates the targeted delivery of methotrexate intercalated montmorillonite clay to cancer cells.

Montmorillonite Clay (MMT) is aimed to develop as an orally administrable drug delivery vehicle with enhanced efficacy. Aiming to enhance the therapeutic index of methotrexate, curcumin is concomitantly used with methotrexate in the present study. Being folate antagonist in nature, methotrexate is internalized into cells by folate receptor (FR); which is over-expressed in certain human cancer cells such as cervical carcinoma cells (HeLa). Firstly, montmorillonite Clay (MMT) is organically modified (OMMT) with cetyl trimethyl ammonium bromide (CTAB) and used to intercalate curcumin and methotrexate separately, designated as OMMT-Cur and OMMT-MTX, respectively. XRD pattern demonstrated successful intercalation of therapeutics and an increase in clay interlayer distance facilitated by CTAB. The dissolution kinetics of methotrexate follows Higuchi model for both Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF), while the release kinetics for curcumin fitted into Higuchi model for SGF and Hixson-Crowell model for SIF, respectively. OMMT-MTX are able to discriminate FR-positive HeLa cells from FR-negative breast cancer cells (MCF7); irrespective of alike cellular phenotypes. Further, the pre-treatment of HeLa cells with curcumin improves its sensitivity towards methotrexate causing a greater killing of the Hela cells. Together, the results propose the concomitant use of curcumin and methotrexate for successfully targeting highly invasive FR-positive carcinomas by means of folate receptor using MMTs.

Chinese Oak Tasar Silkworm Antheraea pernyi Silk Proteins: Current Strategies and Future Perspectives for Biomedical Applications.

Chinese nonmulberry temperate oak tasar/tussah, Antheraea pernyi (Ap) silk is a natural biopolymer that has attracted considerable attention as a biomaterial. The proteinaceous components of Ap silk proteins, namely fibroin and sericin may represent an alternative over mulberry Bombyx mori silk proteins. In fact, the silk fibroin (SF) of Ap is rich in Arginyl-Glycyl-Aspartic acid (RGD) peptides, which facilitate the adhesion and proliferation of various cell types. The possibility of processing Ap silk proteins into different distinct 2D- and 3D-based matrices is described in earlier studies, such as membranes, nanofibers, scaffolds, and micro/nanoparticles, contributing to a different rate of degradation, mechanical properties, and biological performance useful for various biomedical applications. This review summarizes the current advances and developments on nonmulberry Chinese oak tasar silk protein (fibroin and sericin)-based biomaterials and their potential uses in tissue engineering, regenerative medicine, and therapeutic delivery strategies.

Silk fibroin/collagen protein hybrid cell-encapsulating hydrogels with tunable gelation and improved physical and biological properties.

Cell encapsulating hydrogels with tunable mechanical and biological properties are of special importance for cell delivery and tissue engineering. Silk fibroin and collagen, two typical important biological proteins, are considered potential as cell culture hydrogels. However, both have been used individually, with limited properties (e.g., collagen has poor mechanical properties and cell-mediated shrinkage, and silk fibroin from Bombyx mori (mulberry) lacks cell adhesion motifs). Therefore, the combination of them is considered to achieve improved mechanical and biological properties with respect to individual hydrogels. Here, we show that the cell-encapsulating hydrogels of mulberry silk fibroin / collagen are implementable over a wide range of compositions, enabled simply by combining the different gelation mechanisms. Not only the gelation reaction but also the structural characteristics, consequently, the mechanical properties and cellular behaviors are accelerated significantly by the silk fibroin / collagen hybrid hydrogel approach. Of note, the mechanical and biological properties are tunable to represent the combined merits of individual proteins. The shear storage modulus is tailored to range from 0.1 to 20 kPa along the iso-compositional line, which is considered to cover the matrix stiffness of soft-to-hard tissues. In particular, the silk fibroin / collagen hydrogels are highly elastic, exhibiting excellent resistance to permanent deformation under different modes of stress; without being collapsed or water-squeezed out (vs. not possible in individual proteins) - which results from the mechanical synergism of interpenetrating networks of both proteins. Furthermore, the role of collagen protein component in the hybrid hydrogels provides adhesive sites to cells, stimulating anchorage and spreading significantly with respect to mulberry silk fibroin gel, which lacks cell adhesion motifs. The silk fibroin / collagen hydrogels can encapsulate cells while preserving the viability and growth over a long 3D culture period. Our findings demonstrate that the silk / collagen hydrogels possess physical and biological properties tunable and significantly improved (vs. the individual protein gels), implying their potential uses for cell delivery and tissue engineering. STATEMENT OF SIGNIFICANCE: Development of cell encapsulating hydrogels with excellent physical and biological properties is important for the cell delivery and cell-based tissue engineering. Here we communicate for the first time the novel protein composite hydrogels comprised of 'Silk' and 'Collagen' and report their outstanding physical, mechanical and biological properties that are not readily achievable with individual protein hydrogels. The properties include i) gelation accelerated over a wide range of compositions, ii) stiffness levels covering 0.1 kPa to 20 kPa that mimic those of soft-to-hard tissues, iii) excellent elastic behaviors under various stress modes (bending, twisting, stretching, and compression), iv) high resistance to cell-mediated gel contraction, v) rapid anchorage and spreading of cells, and vi) cell encapsulation ability with a long-term survivability. These results come from the synergism of individual proteins of alpha-helix and beta-sheet structured networks. We consider the current elastic cell-encapsulating hydrogels of silk-collagen can be potentially useful for the cell delivery and tissue engineering in a wide spectrum of soft-to-hard tissues.

Emerging tumor spheroids technologies for 3D in vitro cancer modeling.

Cancer is a leading cause of mortality and morbidity worldwide. Around 90% of deaths are caused by metastasis and just 10% by primary tumor. The advancement of treatment approaches is not at the same rhythm of the disease; making cancer a focal target of biomedical research. To enhance the understanding and prompts the therapeutic delivery; concepts of tissue engineering are applied in the development of in vitro models that can bridge between 2D cell culture and animal models, mimicking tissue microenvironment. Tumor spheroid represents highly suitable 3D organoid-like framework elucidating the intra and inter cellular signaling of cancer, like that formed in physiological niche. However, spheroids are of limited value in studying critical biological phenomenon such as tumor-stroma interactions involving extra cellular matrix or immune system. Therefore, a compelling need of tailoring spheroid technologies with physiologically relevant biomaterials or in silico models, is ever emerging. The diagnostic and prognostic role of spheroids rearrangements within biomaterials or microfluidic channel is indicative of patient management; particularly for the decision of targeted therapy. Fragmented information on available in vitro spheroid models and lack of critical analysis on transformation aspects of these strategies; pushes the urge to comprehensively overview the recent technological advancements (e.g. bioprinting, micro-fluidic technologies or use of biomaterials to attain the third dimension) in the shed of translationable cancer research. In present article, relationships between current models and their possible exploitation in clinical success is explored with the highlight of existing challenges in defining therapeutic targets and screening of drug efficacy.

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.

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.

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.

Cytotoxicity and sustained release of modified divinylsulfone from silk based 3D construct.

Monovinylsulfones have been extensively studied for its biological activities but modified divinylsulfones (mDVS2) were largely neglected due to the non-availability of appropriate synthetic routes. The present report describes the potential of a unique derivative of divinylsulfone as a remedial molecule. The mDVS2, available in reasonably large amount through an easy synthesis route, incites necrosis in invasive and non-invasive breast cancer cells in a time and concentration dependent manner. This molecule is further used to fabricate mDVS2 embedded silk based 3D scaffolds in order to achieve sustained release. The entrapped molecules retain their activity over time, as 100% cell death is observed within 7 days. The findings demonstrate the cytotoxic property of mDVS and highlight the importance of under utilized mDVSs as potential therapeutic agents.

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.

Nonmulberry Silk Fibroin Scaffold Shows Superior Osteoconductivity Than Mulberry Silk Fibroin in Calvarial Bone Regeneration.

Recent years have witnessed the advancement of silk biomaterials in bone tissue engineering, although clinical application of the same is still in its infancy. In this study, the potential of pure nonmulberry Antheraea mylitta (Am) fibroin scaffold, without preloading with bone precursor cells, to repair calvarial bone defect in a rat model is explored and compared with its mulberry counterpart Bombyx mori (Bm) silk fibroin. After 3 months of implantation, Am scaffold culminates in a completely ossified regeneration with a progressive increase in mineralization at the implanted site. On the other hand, the Bm scaffold fails to repair the damaged bone, presumably due to its low osteoconductivity and early degradation. The deposition of bone matrix on scaffolds is evaluated by scanning electron and atomic force microscopy. These results are corroborated by in vitro studies of enzymatic degradation, colony formation, and secondary conformational features of the scaffold materials. The greater biocompatibility and mineralization in pure nonmulberry fibroin scaffolds warrants the use of these scaffolds as an "ideal bone graft" biomaterial for effective repair of critical size defects.

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 ß-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.

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.

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.

Thromboelastometric and platelet responses to silk biomaterials.

Silkworm's silk is natural biopolymer with unique properties including mechanical robustness, all aqueous base processing and ease in fabrication into different multifunctional templates. Additionally, the nonmulberry silks have cell adhesion promoting tri-peptide (RGD) sequences, which make it an immensely potential platform for regenerative medicine. The compatibility of nonmulberry silk with human blood is still elusive; thereby, restricts its further application as implants. The present study, therefore, evaluate the haematocompatibility of silk biomaterials in terms of platelet interaction after exposure to nonmulberry silk of Antheraea mylitta using thromboelastometry (ROTEM). The mulberry silk of Bombyx mori and clinically used Uni-Graft W biomaterial serve as references. Shortened clotting time, clot formation times as well as enhanced clot strength indicate the platelet mediated activation of blood coagulation cascade by tested biomaterials; which is comparable to controls.

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.