No abstract

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.

Chronic cutaneous ulcers, a complex pathophysiological diabetic condition, represent a critical clinical challenge in the current diabetes mellitus pandemic. Consequently, there is a compelling need for bioactive dressings that can trigger healing processes for complete wound repair. Silk fibroin (SF), a natural protein polymer from mulberry and non-mulberry silkworms, has properties that support accelerated wound healing rate. SF from non-mulberry variety possesses additional cell-binding motifs (arginine, glycine, and aspartate), offering cell-material interactions. This study is aimed to investigate wound healing efficacy of dressings made up of various SF varieties blended with poly(vinyl alcohol) biopolymer in alloxan-induced diabetic rabbit model. The nanofibrous mats have been developed using electrospinning and functionalized with growth factors and LL-37 antimicrobial peptide for sustained delivery. Following post 14-day treatment, non-mulberry SF (NMSF)-based dressings healed the wounds faster, in comparison with their mulberry Bombyx mori SF, poly(vinyl alcohol), and control counterparts (p < .01). NMSF-based dressings also supported faster granulation tissue development, angiogenesis, and reepithelialization of wounds. Gene expression study of matrix metalloproteinases and collagen proteins affirmed higher extent of tissue remodelling during the repair process. Furthermore, there was organized extracellular matrix deposition (collagen type I, collagen type III, elastin, and reticulin) and higher wound breaking strength in NMSF compared with other groups after 4 weeks. These results validated the potential of NMSF-based bioactive dressings to regulate extracellular matrix deposition leading to faster and complete repair of chronic diabetic cutaneous wounds.

Bioengineering an in vitro liver model recapitulating the native liver microarchitecture consisting of parenchymal and non-parenchymal cells is crucial in achieving cellular crosstalk and hepatic metabolic functions for accurate hepatotoxicity prediction. Bioprinting holds the promise of engineering constructs with precise control over the spatial distribution of multiple cells. Two distinct tissue-specific liver extracellular matrix (ECM)-based bioinks with excellent printability and rheological attributes are formulated for supporting parenchymal and non-parenchymal cells. A physiologically relevant human vascularized liver model is bioprinted with a novel liver ECM-based bioink laden with human adipose mesenchymal stem cell-derived hepatocyte-like cells (HLCs), human umbilical vein endothelial cells (HUVECs), and human hepatic stellate cells (HHSCs) using an extrusion-based bioprinting approach and validated for hepatotoxicity assessment. The HLC/HUVEC/HHSCladen liver model resembles native alternate cords of hepatocytes with a functional sinusoidal lumen-like network in both horizontal and vertical directions, demonstrating enhanced albumin production, urea synthesis, and cytochrome P450 (CPR) activity. Furthermore, the liver model is evaluated for drug toxicity assessment following 24 h exposure to different concentrations of (i) non-hepatotoxicants aspirin and dexamethasone, (ii) idiosyncratic hepatotoxicant trovafloxacin mesylate, and (iii) clinical hepatotoxicant acetaminophen and troglitazone. A followup cell viability and metabolic competence evaluation by estimating DNA concentration, lactate dehydrogenase activity, and CPR activity revealed a dose-dependent clinically relevant hepatotoxic response. These results corroborated that the developed clinically relevant vascularized liver model is affordable and would aid pharmaceutical companies in speeding up the drug development and provide a robust platform for hepatotoxicity screening.

Mimicking nativelike metabolic zonation is indispensable to develop an efficient bioartificial liver model, as it facilitates physiological cues, hepatocyte polarity, and phenotypic functions. The present study shows the first evidence of hepatocyte metabolic heterogeneity in an in vitro liver model encompassing liver extracellular matrix (ECM)-functionalized silk scaffolds (LECM-SF) by altering ECM proportion. Upon static culture, individual LECM-SF scaffold supports differential synthetic and metabolic functions of cultured primary neonatal rat hepatocytes (PNRHs), owing to discrete biophysical attributes. A single in vitro liver system comprising PNRHs seeded LECM-SF scaffolds assisting periportal to pericentral gradient functions is stacked and matured in a perfusion bioreactor to simulate oxygen gradient. The scaffold with high ECM supports periportal-specific albumin synthesis, urea secretion, and bile duct formation, albeit scaffold with low ECM supports pericentral-specific cytochrome P450 activity. Extensive physicochemical characterizations confirmed the stability and interconnected porous network of scaffolds, signifying cellular infiltration and bidirectional nutrient diffusion. Furthermore, scaffolds demonstrate minimal thrombogenicity, reduced foreign-body response, and enhanced pro-remodeling macrophage activation, supporting constructive tissue remodeling. The developed liver model with zone-specific functions would be a promising avenue in bioartificial liver and drug screening.

A hostile myocardial microenvironment post ischemic injury (myocardial infarction) plays a decisive role in determining the fate of tissue-engineered approaches. Therefore, engineering hybrid 3D printed platforms that can modulate the MI microenvironment for improving implant acceptance has surfaced as a critical requirement for reconstructing an infarcted heart. Here, we have employed a non-mulberry silk-based conductive bioink comprising carbon nanotubes (CNTs) to bioprint functional 3D vascularized anisotropic cardiac constructs. Immunofluorescence staining, polymerase chain reaction-based gene expression studies, and electrophysiological studies showed that the inclusion of CNTs in the bioink played a significant role in upregulating matured cardiac biomarkers, sarcomere formation, and beating rate while promoting cardiomyocyte viability. These constructs were then microinjected with calcium peroxide and IL-10-loaded gelatin methacryloyl microspheres. Measurements of oxygen concentration revealed that these microspheres upheld the oxygen availability for maintaining cellular viability for at least 5 days in a hypoxic environment. Also, the ability of microinjected IL-10 microspheres to modulate the macrophages to anti-inflammatory M2 phenotype in vitro was uncovered using immunofluorescent staining and gene expression studies. Furthermore, in vivo subcutaneous implantation of microsphere-injected 3D constructs provided insights toward the extended time frame that was achieved for dealing with the hostile microenvironment for promoting host neovascularization and implant acceptance.

The study of breast cancer metastasis is limited due to poor knowledge of molecular progression of breast tumor and varied heterogeneity. For a better understanding of tumor metastasis, a reliable 3D in vitro model bridging the gap between 2D cultures and in vivo animal model studies is essential. Our study is focused on two key points: (i) designing a 3D microenvironment for studying metastasis and (ii) simulating the metastasis milieu by inducing epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). An electrospun gelatin nanofiber matrix (EGNF) was fabricated using electrospinning and further dip coated with different concentrations of collagen to obtain surface complexity and mechanical properties, similar to connective tissues. Nanofiber matrices were physically characterized by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and field-emission scanning electron microscopy (FESEM). The FTIR, AFM, and FESEM results indicated the crosslinking and confirmed the presence of pores in the nanofiber matrices. Comparative studies on biocompatibility, cell attachment, and the proliferation of MCF-7 cells on EGNF and collagen coated gelatin nanofibrous matrix (CCGM) revealed higher cellular attachment and proliferation in CCGM. CCGM with human metastatic breast cancer cell line (MCF-7) was taken to study breast cancer metastasis using estrogen (induces EMT) and progesterone (induces MET) hormones for 24 h. Quantitative real-time PCR was used for quantifying the expression of metastasis related genes, and fluorescence microscopy for verifying the invasion of cells to the matrices. The expression of E-cadherin and matrix metalloproteinase 2 (MMP 2) confirmed the occurrence of EMT and MET. Live cell imaging and cellular attachment showed significant increase of cellular invasion in crosslinked 0.15% CCGM that serves as a suitable non-toxic, biocompatible, and affordable scaffold for studying breast cancer metastasis. Our findings suggested that CCGM can be used as a tissue-like 3D model for studying breast cancer metastatic events in vitro.

Effective hemorrhage control is indispensable for life-threatening emergencies in defense fields and civilian trauma. During major injuries, hemostatic agents are applied externally to mimic and accelerate the natural hemostasis process. Commercially available topical hemostatic agents are associated with several limitations, e.g., burning sensation, necrosis, futile in severe injuries, and high costs of the products. In the present study, we developed silk fibroin fiber-based formulations and evaluated their use as a cost-effective potential hemostatic agent with shortened clotting time. Silk fiber-based powder was produced following the alkaline hydrolysis process, wherein Bombyx mori silk fibroin fibers were treated with sodium hydroxide (NaOH) solution that randomly chopped the silk microfibers. Physicochemical reaction parameters, e.g., reaction temperature, molarity of NaOH solution, and incubation time, were optimized to achieve the maximum yield of microfibers. The surface properties of alkaline hydrolyzed silk microfibers (AHSMf) were analyzed by field emission scanning electron microscopy and energy dispersive X-ray studies. The water uptake capacity of AHSMf and the change in pH and temperature (∼30 °C) during blood clotting were analyzed. Further, the hemostatic potential of AHSMf was evaluated by an in vitro whole blood clotting assay using both goat and human blood. The in vitro studies demonstrated a reduced blood clotting time (CT = 20–30 s), prothrombin time (PT = ∼27%), and activated partial thromboplastin time (APTT = ∼14%) in the presence of AHSMf when compared to silk hydrogel powder (devoid of NaOH). Thus, the developed AHSMf could be a promising material to serve as a potential hemostatic agent.