Injectable anti-cancer drug loaded silk-based hydrogel for the prevention of cancer recurrence and post-lumpectomy tissue regeneration aiding triple-negative breast cancer therapy.

A single system capable of delivering anticancer drugs and growth factors by a minimally invasive approach is in demand for effective treatment of triple-negative breast cancer (TNBC) after lumpectomy. Here, we showcase one such holistic system for TNBC therapy and its assessment via 3D in vitro lumpectomy model, a first of its kind. Firstly, Bombyx mori silk fibroin (BMSF) and Antheraea assamensis silk fibroin (AASF) blended hydrogels were prepared and biophysically characterized. Secondly, a 3D in vitro lumpectomy model was developed using MDA-MB-231 cell line to assess the efficacy of localized delivery of doxorubicin (dox) using injectable hydrogel system in terminating remaining breast cancer after lumpectomy. Additionally, we have also evaluated the adipose tissue regeneration in the lumpectomy region by delivering dexamethasone (dex) using injectable hydrogels. Rheological studies showed that the BMSF/AASF blended hydrogels exhibit viscoelasticity and injectability conducive for minimally invasive application. The developed hydrogels by virtue of its slow and sustained release of dox exerted cytotoxicity towards MDA-MB-231 cells assessed through in vitro studies. Further, dex loaded hydrogel supported adipogenic differentiation of adipose tissue derived stem cells (ADSCs), while the secreted factors were found to aid in vascularization and macrophage polarization. This was confirmed through in vitro angiogenic tube formation assay and macrophage polarization study respectively. The corroborated results vouch for potential application of this injectable hydrogels for localized anticancer drug delivery and aiding in breast reconstruction, post lumpectomy.

3D Bioprinted Silk-Based In Vitro Osteochondral Model for Osteoarthritis Therapeutics.

3D bioprinting of osteochondral tissue offers unique opportunities for enabling precise pharmacological interventions in osteoarthritis (OA). The current study investigates the screening potential of anti-inflammatory drugs using bioprinted inflamed human osteochondral units. The biomimetic hierarchical geometry is bioprinted using silk-based bioinks encapsulating pre-differentiated stem cells, creating an in vitro model. Inflammation is stimulated in the model, using tumor necrosis factor-alpha and Interleukin-1 beta pro-inflammatory cytokines. The resultant degeneration, akin to OA, is flagged by key markers like sulfated glycosaminoglycan, collagen, alkaline phosphatase, and downregulation of osteochondral transcript levels. In the next step, the screening of anti-inflammatory drugs is validated using celecoxib and rhein. Consequently, in the inflamed constructs, the initial upregulation of the key inflammatory mediators (nitric oxide, Prostaglandin E2), is subsequently downregulated, post-drug treatment. In addition, catabolic markers (matrix metalloproteinases and aggrecanase-1), indicative of hypertrophic and apoptosing chondrocytes, are significantly downregulated in the treatment groups; while the transcript and protein levels required for osteochondral health are attenuated. Therefore, the in vitro model mimicks the inflammation in the early stages of OA, and corroborates a potential high-throughput platform for screening novel anti-inflammatory drugs in OA therapeutics.

Mesoporous Silk-Bioactive Glass Nanocomposites as Drug Eluting Multifunctional Conformal Coatings for Improving Osseointegration and Bactericidal Properties of Metal Implants.

Endowing metal implants with multifunctional traits to prevent implant-associated infections and improve osseointegration has become a pivotal facet in orthopedics and dental fixation. Herein, we report the synthesis of mesoporous 70S bioactive glass-silk fibroin nanocomposites inspired by the biomimetic organo-apatites of mineralized collagen. The mesoporous, biomimetic nanocomposites enabled loading of antibiotics (gentamicin and doxycycline) and favored their release in a rapid manner while preserving their bioactivity. Ease in modification of the mesoporous nanocomposites enabled tailoring of 3-(aminopropyl)-triethoxysilane to the silanol network of bioactive glass, which improved the loading capacity of the hydrophobic drug (dexamethasone). The modification favored the slow and sustained release of dexamethasone from the modified mesoporous nanocomposites, which is desired for mediating osteogenesis and immunomodulation. Conformal coatings of these drug-loaded nanocomposites were materialized on stainless-steel implants through a facile electrophoretic deposition (EPD) technique, wherein the deposition yield can be controlled by applied parameters. Antibiotic coatings exhibited antibacterial efficacy with bioactivity retained up to 28 days, while dexamethasone-loaded coatings favored mesenchymal stem cell adhesion and osteoinduction. The immunomodulatory roles were also ascertained, wherein M2 macrophage biasness was favored in dexamethasone-loaded coatings. The versatility of these mesoporous biomimetic nanocomposites guarantee the loading of scenario-specific drugs to aid their local delivery through the conformal EPD coatings developed over metal implants toward improving implant patency.

Silk-Based Bioengineered Diaphyseal Cortical Bone Unit Enclosing an Implantable Bone Marrow toward Atrophic Nonunion Grafting.

Postnatal fracture healing of atrophic long bone diaphyseal nonunions remains a challenge for orthopedic surgeons. Paucity of autologous spongiosa has potentiated the use of tissue engineered bone grafts to improve success rates of bone marrow engraftment used in plate reosteosynthesis. Herein, the development and in vitro validation of a "sandwich-type" biofabricated diaphyseal cross-sectional unit, with an outer mechanically robust bioprinted cortical bone shell, encompassing an engineered bone marrow, are reported. Channelized silk fibroin blend sponges derived from Bombyx mori and Antheraea assama help in developing compartmentalized endosteum, exhibiting specialized osteoblasts (endosteal niche) and discontinuous endothelium (vascular niche). The cellular cross-talk between these two niches triggered via integrin-mediated cell adhesion, enables in preserving quiescence state of CD34+ /CD38- hematopoietic stem cells and their recycling in the engineered marrow. The outer cortical bone strut is developed through multimaterial microextrusion bioprinting strategy. Osteogenically primed mesenchymal stem cells-laden silk fibroin-nano-hydroxyapatite bioink is bioprinted alongside paramagnetic Fe-doped bioactive glass-polycaprolactone blend thermoplastic ink, reinforcing it for mechanical stability. Pulsed magnetic field actuation positively influences the osteogenic commitment and maturation of the bioprinted constructs via mechanotransductory route. Therefore, the assembled engineered marrow and bioprinted cortical shell hold promise as potential orthobiologic substitutes toward atrophic nonunion repairs.

Overcoming the Dependence on Animal Models for Osteoarthritis Therapeutics - The Promises and Prospects of In Vitro Models.

Osteoarthritis (OA) is a musculoskeletal disease characterized by progressive degeneration of osteochondral tissues. Current treatment is restricted to the reduction of pain and loss of function of the joint. To better comprehend the OA pathophysiological conditions, several models are employed, however; there is no consensus on a suitable model. In this review, different in vitro models being developed for possible therapeutic intervention of OA are outlined. Herein, various in vitro OA models starting from 2D model, co-culture model, 3D models, dynamic culture model to advanced technologies-based models such as 3D bioprinting, bioassembly, organoids, and organ-on-chip-based models are discussed with their advantages and disadvantages. Besides, different growth factors, cytokines, and chemicals being utilized for induction of OA condition are reviewed in detail. Furthermore, there is focus on scrutinizing different molecular and possible therapeutic targets for better understanding the mechanisms and OA therapeutics. Finally, the underlying challenges associated with in vitro models are discussed followed by future prospective. Taken together, a comprehensive overview of in vitro OA models, factors to induce OA-like conditions, and intricate molecular targets with the potential to develop personalized osteoarthritis therapeutics in the future with clinical translation is provided.

Unconventional and Facile Fabrication of Chemically Reactive Silk Fibroin Sponges for Environmental Remediation.

Silk fibroin and silk microfibers, both derived from silk cocoon, have been widely used for prospective biomedical, energy, and environmental applications. However, various complex and catalyst-based approaches have been adopted for chemical modification and integration of different functionalities in silk fibroin-based materials. Here, both tailored water wettability and mechanical property have been associated with silk microfiber reinforced silk fibroin sponges (SMFRSFSs) through the strategic introduction of ß-sheets and a facile and catalyst-free chemical reaction at ambient conditions. While the controlled tailoring of ß-sheets in the silk fibroin skeletal framework of the sponges allowed us to modulate the compressive modulus, the 1,4-conjugate addition reaction between amine residues of silk (fiber and fibroin) and acrylate groups of a multifunctional cross-linker provided residual chemical reactivity. Further, the chemically "reactive" sponge was postmodified with the selected alkylamines to introduce a wide range of water wettability (from 36 to 161°) without affecting the mechanical property. Thereafter, the silk cocoon-derived and extremely water-repellent sponge was used for environment-friendly cleaning of oil spillages through selective absorption-based and filtration-based oil/water separation at different and severe aqueous conditions. This silk cocoon-derived mechanically tailorable and chemically reactive sponge could also be useful for various biomedical and energy-related applications.

Insight into Silk-Based Biomaterials: From Physicochemical Attributes to Recent Biomedical Applications.

Silk, a natural biopolymer, has been used clinically as suture material over thousands of years and has received much impetus for a plethora of biomedical applications in the last two decades. Silk protein isolated from both mulberry and nonmulberry silkworm varieties gained recognition as a potential biomaterial owing to its affordability and remarkable physicochemical properties. Molecular studies on the amino acid composition and conformation of silk proteins interpreted in the present review provide a critical understanding of the difference in crystallinity, hydrophobicity, and tensile strength among silkworm silk proteins. Meticulous silk fibroin (SF) isolation procedures and innovative processing techniques to fabricate gamut of two-dimensional (2D) and three-dimensional (3D) matrices including the latest 3D printed scaffolds have led SF for diverse biomedical applications. Crucial factors for clinical success of any biomaterial, including biocompatibility, immune response, and biodegradability, are discussed with particular emphasis on the lesser-known endemic nonmulberry silk varieties, which in recent years have gained considerable attention. The tunable biodegradation and bioresorbable attributes of SF enabled its use in drug delivery systems, thus proving it as an efficient and specific vehicle for controlled drug release and targeted drug delivery. Advancements in fabrication methodologies inspired biomedical researchers to develop SF-based in vitro tissue models mimicking the spatiotemporal arrangement and cellular distribution of native tissue. In vitro tissue models own a unique demand for studying tissue biology, cellular crosstalks, disease modeling, drug designing, and high throughput drug screening applications. Significant progress in silk biomaterial research has evolved into several silk-based healthcare products in the market. Insights of silk-based products assessed in the human clinical trials are presented in this review. Overall, the current review explores the paradigm of the silk structure-function relationship driving silk-based biomaterials toward tissue engineering, drug delivery systems, and in vitro tissue models.

Synergistic Effects of Silicon/Zinc Doped Brushite and Silk Scaffolding in Augmenting the Osteogenic and Angiogenic Potential of Composite Biomimetic Bone Grafts.

Cell instructive scaffolding platforms displaying synergistic effects by virtue of their chemical and physical cues have tremendous scope in modulating cell phenotype and thus improving the success of any graft. In this regard, we report here the development of Si- and Zn-doped brushite cement composited with silk scaffolding that hierarchically emulated the cancellous bone. The composite scaffolds fabricated exhibited an open porous network capable of enhanced osteoblast survival as attested by increased alkaline phosphatase activity and also sustaining osteoclast activity affirmed by tartrate resistant acid phosphatase staining. Moreover, the chemical cues presented by dissolutions products from the composite scaffold enabled the osteoblasts to secrete proangiogenic factors which favored better endothelial cell survival, confirmed through in vitro experiments. Moreover, the efficacy of these composite biomimetic scaffolds was validated in vivo in volumetric femur defects in rabbits, which revealed that these matrices influenced vascular cell infiltration and favored the formation of matured bony plate. Fluorochrome labeling studies and microtomography analysis revealed that at the end of three months, the implanted composite scaffolds had completely resorbed, leaving behind neo-osseous tissue and vouching for clinical translation of these composite matrices as viable and affordable bone-graft substitutes.

Surface Patterning and Innate Physicochemical Attributes of Silk Films Concomitantly Govern Vascular Cell Dynamics.

Functional impairment of vascular cells is associated with cardiovascular pathologies. Recent literature clearly presents evidence relating cell microenvironment and their function. It is crucial to understand the cell-material interaction while designing a functional tissue engineered vascular graft. Natural silk biopolymer has shown potential for various tissue-engineering applications. In the present work, we aimed to explore the combinatorial effect of variable innate physicochemical properties and topographies of silk films on functional behavior of vascular cells. Silk proteins from different varieties (mulberry Bombyx mori, BM; and non-mulberry Antheraea assama, AA) possess unique inherent amino acid composition that leads to variable surface properties (roughness, wettability, chemistry, and mechanical stiffness). In addition, we engineered the silk film surfaces and printed a microgrooved pattern to induce unidirectional cell orientation mimicking their native form. Patterned silk films induced unidirectional alignment of porcine vascular cells. Regardless of alignment, endothelial cells (ECs) proliferated favorably on AA films; however, it suppressed production of nitric oxide (NO), an endogenous vasodilator. Unidirectional alignment of smooth muscle cells (SMCs) encouraged contractile phenotype as indicated by minimal cell proliferation, increment of quiescent (G0) phase cells, and upregulation of contractile genes. Moderately hydrophilic flat BM films induced cell aggregation and augmented the expression of contractile genes (for SMCs) and endothelial nitric oxide synthase, eNOS (for ECs). Functional studies further confirmed SMCs' alignment improving collagen production, remodeling ability (matrix metalloproteinase, MMP-2 and MMP-9 production) and physical contraction. Altogether, this study confirms vascular cells' functional behavior is crucially regulated by synergistic effect of their alignment and cell-substrate interfacial properties.

3D Printing/Bioprinting Based Tailoring of in Vitro Tissue Models: Recent Advances and Challenges.

Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed.

Multifunctional Cell Instructive Silk-Bioactive Glass Composite Reinforced Scaffolds Toward Osteoinductive, Proangiogenic, and Resorbable Bone Grafts.

The successful regeneration of large volume bone defects necessitates the use of proangiogenic and resorbable scaffolding matrix. Impaired and slow ingrowth of host vasculature within implanted grafts greatly compromises its effective osseointegration. By addressing this, it is demonstrated that the use of copper doped bioactive glass functionalizes silk microfiber reinforcements to improve the physicochemical and osteoinductive properties of two silk scaffolding matrices (mulberry Bombyx mori and non-mulberry Antheraea assama) employed in the study. The reinforced composite matrices increase the surface area and present an open porous biomimetic micromillieu favoring stem cell and endothelial cell migration within the matrix. Biochemical results indicate the stabilization of hypoxia-inducible factor-1α and expression of C-X-C chemokine receptor type-4 in adipose derived human mesenchymal stem cells, which regulate the downstream proangiogenic signaling and endothelial cell homing, respectively. Osteoinduction, matrix turnover, and resorption effectiveness are favored better in the non-mulberry silk matrices. The composite matrices significantly promote neo-osseous tissue formation in volumetric femur defect in rabbits with periosteal restoration seen in the non-mulberry silk composite matrices. Evidences of total resorption, enhanced vascular-fibrous tissue ingrowth within the scaffold, vouch for the potential clinical translation of these developed composite silk matrices.

Injectable hydrogels: a new paradigm for osteochondral tissue engineering.

Osteochondral tissue engineering has become a promising strategy for repairing focal chondral lesions and early osteoarthritis (OA), which account for progressive joint pain and disability in millions of people worldwide. Towards improving osteochondral tissue repair, injectable hydrogels have emerged as promising matrices due to their wider range of properties such as their high water content and porous framework, similarity to the natural extracellular matrix (ECM), ability to encapsulate cells within the matrix and ability to provide biological cues for cellular differentiation. Further, their properties such as those that facilitate minimally invasive deployment or delivery, and their ability to repair geometrically complex irregular defects have been critical for their success. In this review, we provide an overview of innovative approaches to engineer injectable hydrogels towards improved osteochondral tissue repair. Herein, we focus on understanding the biology of osteochondral tissue and osteoarthritis along with the need for injectable hydrogels in osteochondral tissue engineering. Furthermore, we discuss in detail different biomaterials (natural and synthetic) and various advanced fabrication methods being employed for the development of injectable hydrogels in osteochondral repair. In addition, in vitro and in vivo applications of developed injectable hydrogels for osteochondral tissue engineering are also reviewed. Finally, conclusions and future perspectives of using injectable hydrogels in osteochondral tissue engineering are provided.

Hierarchically structured seamless silk scaffolds for osteochondral interface tissue engineering.

The osteochondral healthcare market is driven by the increasing demand for affordable and biomimetic scaffolds. To meet this demand, silk fibroin (SF) from Bombyx mori and Antheraea assamensis is used to fabricate a biphasic scaffold, with fiber-free and fiber-reinforced phases, stimulating cartilage and bone revival. The fabrication is a facile reproducible process using single polymer (SF), for both phases, designed in a continuous and integrated manner. Physicochemical and mechanical scaffold characterization, display interconnected pores with differential swelling and tunable degradation. The compressive modulus values, extend to 40 kPa and 25%, for tensile strain, at elongation. The scaffold support, for growth and proliferation of chondrocytes and osteoblasts, for respective cartilage and bone regeneration, is verified from in vitro assessment. Up-regulation of alkaline phosphatase (ALP) activity, extracellular matrix secretion and gene expression are significant; with acceptable in vitro immune response. Upon implantation in rabbit osteochondral defects for 8 weeks, the histological and micro-CT examinations show biphasic scaffolds significantly enhance regeneration of cartilage and subchondral bone tissues, as compared to monophasic scaffolds. The regenerated bone mineral density (BMD) ranges from 600-700 mg hydroxyapatite (HA) per cm3. The results, therefore, showcase the critically positive characteristics of in vitro ECM deposition, and in vivo regeneration of osteochondral tissue by this hierarchically structured biphasic scaffold.