Injectable Self-Oxygenating Cardio-Protective and Tissue Adhesive Silk-Based Hydrogel for Alleviating Ischemia After Mi Injury.

Myocardial infarction (MI) is a significant cardiovascular disease that restricts blood flow, resulting in massive cell death and leading to stiff and noncontractile fibrotic scar tissue formation. Recently, sustained oxygen release in the MI area has shown regeneration ability; however, improving its therapeutic efficiency for regenerative medicine remains challenging. Here, a combinatorial strategy for cardiac repair by developing cardioprotective and oxygenating hybrid hydrogels that locally sustain the release of stromal cell-derived factor-1 alpha (SDF) and oxygen for simultaneous activation of neovascularization at the infarct area is presented. A sustained release of oxygen and SDF from injectable, mechanically robust, and tissue-adhesive silk-based hybrid hydrogels is achieved. Enhanced endothelialization under normoxia and anoxia is observed. Furthermore, there is a marked improvement in vascularization that leads to an increment in cardiomyocyte survival by ≈30% and a reduction of the fibrotic scar formation in an MI animal rodent model. Improved left ventricular systolic and diastolic functions by ≈10% and 20%, respectively, with a ≈25% higher ejection fraction on day 7 are also observed. Therefore, local delivery of therapeutic oxygenating and cardioprotective hydrogels demonstrates beneficial effects on cardiac functional recovery for reparative therapy.

Neoantigen Immunotherapeutic-Gel Combined with TIM-3 Blockade Effectively Restrains Orthotopic Hepatocellular Carcinoma Progression.

Herein, we integrate the Hepa1-6 liver cancer-specific neoantigen, toll-like receptor 9 agonist and stimulator of interferon genes agonist by silk-hydrogel package, and combine with TIM-3 blockade to elicit robust antitumor immunity for effectively suppressing orthotopic hepatocellular carcinoma (HCC) progression. Unlike intradermal injection of simple mixed components with short-term immune protection, the neoantigen immunotherapeutic-gels evoke long-term immune protection to achieve significant prophylactic and therapeutic activity against HCC through only one-shot administration without any side effects. Notably, the synergized immunotherapy by further combining NGC-gels with TIM-3 antibody significantly reduces regulatory T-cells and increases the IFN-γ and IL-12p70 levels in tumor tissues for promoting the infiltration of IFN-γ+CD8+T-cells and 41BB+CD8+T-cells to achieve complete remission (4/7) and prevent pulmonary metastasis in orthotopic HCC, and establish long-term memory against tumor rechallenge with remarkably longer survival time (180 days). Overall, this study provides an attractive and promising synergistic strategy for HCC immunotherapy with possible clinical translation prospects.

A silk-based hydrogel containing dexamethasone and lipoic acid microcrystals for local delivery to the inner ear.

Local drug delivery has been exploited recently to treat hearing loss, as this method can both bypass the blood-labyrinth barrier and provide sustained drug release. Combined drug microcrystals (MCs) offer additional advantages for sensorineural hearing loss treatment via intratympanic (IT) injection due to their shape effect and combination strategy. In this study, to endow viscous effects of hydrogels, nonspherical dexamethasone (DEX) and lipoic acid (LA) MCs were incorporated into silk fibroin (SF) hydrogels, which were subsequently administered to the tympanic cavity to investigate their pharmaceutical properties. First, we prepared DEX and LA MCs by a traditional precipitation technique followed by SF hydrogel incorporation (SF+DEX+LA). After characterization of the physicochemical features, including morphology, rheology, and dissolution, both a suspension of combined DEX and LA MCs (DEX+LA) and SF+DEX+LA were administered to guinea pigs by IT injection, after which the pharmacokinetics, biodegradation and biocompatibility were evaluated. To our surprise, compared to the DEX+LA group, the pharmacokinetics of the SF+DEX+LA hydrogel group did not improve significantly, which may be ascribed to their nonspherical shape and deposition effects of the drugs MCs. The cochlear tissue in each group displayed good morphology, with no obvious inflammatory reactions. This combined MC suspension has the clear advantages of no vehicle, easy scale-up preparation, and good biocompatibility and outcomes, which paves the way for practical treatment of hearing loss via local drug delivery.

Macrophage metabolic reprogramming-based diabetic infected bone defect/bone reconstruction though multi-function silk hydrogel with exosome release.

Diabetic infected bone defects (DIBD) with abnormal immune metabolism are prone to the hard-to-treat bacterial infections and delayed bone regeneration, which present significant challenges in clinic. Control of immune metabolism is believed to be important in regulating fundamental immunological processes. Here, we developed a macrophage metabolic reprogramming hydrogel composed of modified silk fibroin (Silk-6) and poly-l-lysine (ε-PL) and further integrated with M2 Macrophage-derived Exo (M2-Exo), named Silk-6/ε-PL@Exo. This degradable hydrogel showed a broad-spectrum antibacterial performance against both Gram-positive and -negative bacteria. More importantly, the release of M2-Exo from Silk-6/ε-PL@Exo could target M1 macrophages, modulating the activity of the key enzyme hexokinase II (HK2) to control the inflammation-related NF-κB pathway, alleviate lactate accumulation, and inhibit glycolysis to normalize the cycle, thereby promoting M1-to-M2 balance. Using a rat model of DIBD, Silk-6/ε-PL@Exo hydrogel promoted infection control, balanced immune responses and accelerated the bone defect healing. Overall, this study demonstrates that this Silk-6/ε-PL @Exo is a promising filler biomaterial with multi-function to treat DIBD and emphasizes the importance of metabolic reprogramming in bone regeneration.

Self-assembling doxorubicin silk hydrogels for the focal treatment of primary breast cancer.

Standard care for early stage breast cancer includes tumor resection and local radiotherapy to achieve long-term remission. Systemic chemotherapy provides only low locoregional control of the disease; therefore, we describe self-assembling silk hydrogels that can retain and then deliver doxorubicin locally. Self-assembling silk hydrogels show no swelling, are readily loaded with doxorubicin under aqueous conditions and release drug over 4 weeks in amounts that can be fine-tuned by varying the silk content. Following successful in vitro studies, locally injected silk hydrogels loaded with doxorubicin show excellent antitumor response in mice, outperforming the equivalent amount of doxorubicin delivered intravenously. In addition to reducing primary tumor growth, doxorubicin-loaded silk hydrogels reduce metastatic spread and are well tolerated in vivo. Thus, silk hydrogels are well suited for the local delivery of chemotherapy and provide a promising approach to improve locoregional control of breast cancer.

A biomaterial-silicon junction for photodetection.

Bio-integrated optoelectronics can be interfaced with biological tissues, thereby offering opportunities for clinical diagnosis and therapy. However, finding a suitable biomaterial-based semiconductor to interface with electronics is still challenging. In this study, a semiconducting layer is assembled comprising a silk protein hydrogel and melanin nanoparticles (NPs). The silk protein hydrogel provides a water-rich environment for the melanin NPs that maximizes their ionic conductivity and bio-friendliness. An efficient photodetector is produced by forming a junction between melanin NP-silk and a p-type Si (p-Si) semiconductor. The observed charge accumulation/transport behavior at the melanin NP-silk/p-Si junction is associated with the ionic conductive state of the melanin NP-silk composite. The melanin NP-silk semiconducting layer is printed as an array on an Si substrate. The photodetector array exhibits uniform photo-response to illumination at various wavelengths, thus providing broadband photodetection. Efficient charge transfer between melanin NP-silk and Si provides fast photo-switching with rise and decay constants of 0.44 â€‹s and 0.19 â€‹s, respectively. The photodetector with a biotic interface comprising an Ag nanowire-incorporated silk layer as the top contact can operate when underneath biological tissue. The photo-responsive biomaterial-Si semiconductor junction using light as a stimulus offers a bio-friendly and versatile platform for artificial electronic skin/tissue.

Fabrication of green composite hand knitted silk mesh reinforced with silk hydrogel.

Soft tissue defects like hernia and post-surgical fistula formation can be resolved with modern biomaterials in the form of meshes without post-operative complications. In the present study hand knitted silk meshes were surface coated with regenerated silk fibroin hydrogel and pure natural extracts. Two phytochemicals (Licorice extract (LE) and Bearberry extract (BE)) and the two honeybee products (royal jelly (RJ) and honey (HE)) were incorporated separately to induce antibacterial, anti-inflammatory, and wound healing ability to the silk hydrogel coated knitted silk meshes. Meshes were dip coated with a blend of 4 % silk hydrogel (w/v) and 5 % extracts. Dried modified meshes were characterized using SEM, DMA, GC-MS and FTIR. Antimicrobial testing, in-vitro cytotoxicity, in-vitro wound healing and Q-RT-PCR were also performed. SEM analysis concluded that presence of coating reduced the pore size up to 47.7 % whereas, fiber diameter was increased up to 17.9 % as compared to the control. The presence of coating on the mesh improved the mechanical strength/Young's modulus by 1602.8 %, UTS by 451.7 % and reduced the % strain by 51.12 %. Sustained release of extracts from MHRJ (62.9 % up to 72 h) confirmed that it can induce antibacterial activity against surgical infections. Cytocompatibility testing and gene expression results suggest that out of four variables MHRJ presented best cell viability, % wound closure and expression of wound healing marker genes. In-vivo analyses in rat hernia model were carried out using only MHRJ variant, which also confirmed the non- toxic nature and wound healing characteristics of the modified mesh. The improved cell proliferation and activated wound healing in vitro and in vivo suggested that MHRJ could be a valuable candidate to promote cell infiltration and activate soft tissue and hernia repair as a biomedical implant.

Fabrication of Silk Hydrogel Scaffolds with Aligned Porous Structures and Tunable Mechanical Properties.

The effectiveness of cell culture and tissue regeneration largely depends on the structural and physiochemical characteristics of tissue-engineering scaffolds. Hydrogels are frequently employed in tissue engineering because of their high-water content and strong biocompatibility, making them the ideal scaffold materials for simulating tissue structures and properties. However, hydrogels created using traditional methods have low mechanical strength and a non-porous structure, which severely restrict their application. Herein, we successfully developed silk fibroin glycidyl methacrylate (SF-GMA) hydrogels with oriented porous structures and substantial toughness through directional freezing (DF) and in situ photo-crosslinking (DF-SF-GMA). The oriented porous structures in the DF-SF-GMA hydrogels were induced by directional ice templates and maintained after photo-crosslinking. The mechanical properties, particularly the toughness, of these scaffolds were enhanced compared to the traditional bulk hydrogels. Interestingly, the DF-SF-GMA hydrogels exhibit fast stress relaxation and variable viscoelasticity. The remarkable biocompatibility of the DF-SF-GMA hydrogels was further demonstrated in cell culture. Accordingly, this work reports a method to prepare tough SF hydrogels with aligned porous structures, which can be extensively applied to cell culture and tissue engineering.

Focal Infection Treatment using Laser-Mediated Heating of Injectable Silk Hydrogels with Gold Nanoparticles.

Medical treatment of subcutaneous bacterial abscesses usually involves systemic high-dose antibiotics and incision-drainage of the wound. Such an approach suffers from two main deficiencies: bacterial resistance to antibiotics and pain associated with multiple incision-drainage-wound packing procedures. Furthermore, the efficacy of high-dose systemic antibiotics is limited because of the inability to penetrate into the abscess. To address these obstacles, we present a treatment relying on laser-induced heating of gold nanoparticles embedded in an injectable silk-protein hydrogel. Although bactericidal nanoparticle systems have been previously employed based on silver and nitric oxide, they have limitations regarding customization and safety. The method we propose is safe and uses biocompatible, highly tunable materials: an injectable silk hydrogel and Au nanoparticles, which are effective absorbers at low laser powers such as those provided by hand held devices. We demonstrate that a single 10-minute laser treatment of a subcutaneous infection in mice preserves the general tissue architecture, while achieving a bactericidal effect - even resulting in complete eradication in some cases. The unique materials platform presented here can provide the basis for an alternative treatment of focal infections.

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure.

Objective: Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete "classical" depots, or interspersed in white adipose tissue (WAT), termed "inducible/recruitable" BAT, or 'beige/brite' adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. Methods: In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for in vivo delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior in vitro brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive in vivo whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Results: Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in "browning", including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). Conclusions: In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments.

Engineered Tough Silk Hydrogels through Assembling ß-Sheet Rich Nanofibers Based on a Solvent Replacement Strategy.

ß-Sheet rich silk nanofiber hydrogels are suitable scaffolds in tissue regeneration and carriers for various drugs. However, unsatisfactory mechanical performance limits its applications. Here, insight into the silk nanofibers stimulates the remodeling of previous solvent systems to actively regulate the assembly of silk nanofibers. Formic acid, a solvent of regenerated silk fibroin, is used to shield the charge repulsion of silk nanofibers to facilitate the nanofiber assembly under concentrated solutions. Formic acid was replaced with water to solidify the assembly, which induced the formation of a tough hydrogel. The hydrogels generated with this process possessed a modulus of 5.88 ± 0.82 MPa, ultimate stress of 1.55 ± 0.06 MPa, and toughness of 0.85 ± 0.03 MJ m-3, superior to those of previous silk hydrogels prepared through complex cross-linking processes. Benefiting from the dense gel network and high ß-sheet content, these silk nanofiber hydrogels had good stability and antiswelling ability. The modulus could be modulated via changing the silk nanofiber concentration to provide differentiation signals to stem cells. Improved mechanical and bioactive properties with these hydrogels suggest utility in biomedical and engineering fields. More importantly, our present study reveals that the in-depth understanding of silk nanofibers could infuse power into traditional fabrication systems to achieve more high performance biomaterials, which is seldom considered in silk material studies.

3D printed silk-gelatin hydrogel scaffold with different porous structure and cell seeding strategy for cartilage regeneration.

Hydrogel scaffolds are attractive for tissue defect repair and reorganization because of their human tissue-like characteristics. However, most hydrogels offer limited cell growth and tissue formation ability due to their submicron- or nano-sized gel networks, which restrict the supply of oxygen, nutrients and inhibit the proliferation and differentiation of encapsulated cells. In recent years, 3D printed hydrogels have shown great potential to overcome this problem by introducing macro-pores within scaffolds. In this study, we fabricated a macroporous hydrogel scaffold through horseradish peroxidase (HRP)-mediated crosslinking of silk fibroin (SF) and tyramine-substituted gelatin (GT) by extrusion-based low-temperature 3D printing. Through physicochemical characterization, we found that this hydrogel has excellent structural stability, suitable mechanical properties, and an adjustable degradation rate, thus satisfying the requirements for cartilage reconstruction. Cell suspension and aggregate seeding methods were developed to assess the inoculation efficiency of the hydrogel. Moreover, the chondrogenic differentiation of stem cells was explored. Stem cells in the hydrogel differentiated into hyaline cartilage when the cell aggregate seeding method was used and into fibrocartilage when the cell suspension was used. Finally, the effect of the hydrogel and stem cells were investigated in a rabbit cartilage defect model. After implantation for 12 and 16 weeks, histological evaluation of the sections was performed. We found that the enzymatic cross-linked and methanol treatment SF5GT15 hydrogel combined with cell aggregates promoted articular cartilage regeneration. In summary, this 3D printed macroporous SF-GT hydrogel combined with stem cell aggregates possesses excellent potential for application in cartilage tissue repair and regeneration.

Exploring Gelation and Physicochemical Behavior of in Situ Bioresponsive Silk Hydrogels for Disc Degeneration Therapy.

Hydrogels have received considerable attention in the field of tissue engineering because of their unique structural and compositional resemblance to the highly hydrated human tissues. In addition, controlled fabrication processes benefit them with desirable physicochemical features for injectability in minimally invasive manner and cell survival within hydrogels. Formulation of biologically active hydrogels with desirable characteristics is one of the prerequisites for successful applications like nucleus pulposus (NP) tissue engineering to address disc degeneration. To achieve such a benchmark, in this study, two naturally derived silk fibroin proteins (Bombyx mori, BM SF; and Antheraea assamensis, AA SF) were blended together to allow self-assembly and transformation to hydrogels in absence of any cross-linker or external stimuli. A comprehensive study on sol-gel transition of fabricated hydrogels in physiological fluid microenvironment (pH, temperature, and ionic strength) was conducted using optical and fluorescence analysis. Tunable gelation time (∼8-40 min) was achieved depending on combinations. The developed hydrogels were validated by extensive physicochemical characterizations which include confirmation of secondary structure, surface morphology, swelling and degradation. Mechanical behavior of the hydrogels was further analyzed in various in vitro-physiological-like conditions with varying pH, ionic strength, diameter, storage time, and strain values to determine their suitability in native physiological environments. Rheological study, cytocompatibility using primary porcine NP cells and ex vivo biomechanics of hydrogels were explored to validate their in situ applicability in minimally invasive manner toward potential disc regeneration therapy.

Injectable Carbon Nanotube Impregnated Silk Based Multifunctional Hydrogel for Localized Targeted and On-Demand Anticancer Drug Delivery.

The major limitations of traditional methods of anticancer drug delivery include systemic distribution and frequent administration intravenously. To address these issues, in our present approach, we have fabricated a nano hybrid silk hydrogel system for localized, targeted, and on-demand delivery of anticancer drugs. The hybrid system contains a blend of two varieties of silk protein and doxorubicin (DOX)-loaded folic acid functionalized single-walled carbon nanotubes (SWCNT-FA/DOX). Owing to the single-walled carbon nanotube (SWCNT) incorporation, the mechanical strength of the hybrid silk hydrogel composite enhanced significantly. A slow and sustained DOX release was recorded over a 14 day study. The amount of DOX released was determined by concentration of the SWCNT-FA/DOX payload, rate of silk degradation, pH of the released medium, and incubation temperature. The intermittent exposure of near-infrared light to the hybrid gel system stimulated on-demand DOX release. The in vitro studies demonstrated the active targeting of SWCNT-FA/DOX to folic acid receptor-positive (FR+ve) cancer cells. The silk hydrogel, being viscoelastic in nature, is easily injectable to the targeted site. Hence, the developed silk hybrid gel system may allow its near or intratumoral implantation, where it may act as a depot for anticancer drug-loaded nanoparticles. The sustained, targeted, and external-stimuli-dependent DOX released at the localized tumor site is expected to reduce its systemic side effects and show an efficient way to treat the cancer.

Silk fibroin hydrogel as physical barrier for prevention of post hernia adhesion.

BACKGROUND: Adhesion formation remains a major complication following hernia repair surgery. Physical barriers though effective for adhesion prevention in clinical settings are associated with major disadvantages, therefore, needs further investigation. This study evaluates silk fibroin hydrogel as a physical barrier on polypropylene mesh for the prevention of adhesion following ventral hernia repair. STUDY DESIGN: Peritoneal explants were cultured on silk fibroin scaffold to evaluate its support for mesothelial cell growth. Full thickness uniform sized defects were created on the ventral abdominal wall of rabbits, and the defects were covered either with silk hydrogel coated polypropylene mesh or with plain polypropylene mesh as a control. The animals were killed after 1 month, and the adhesion formation was graded; healing response of peritoneum was evaluated by immunohistochemistry with calretinin, collagen staining of peritoneal sections, and expression of PCNA, collagen-I, TNFα, IL6 by real time PCR; and its adverse effect if any was determined. RESULTS: Silk fibroin scaffold showed excellent support for peritoneal cell growth in vitro and the cells expressed calretinin. A remarkable prevention of adhesion formation was observed in the animals implanted with silk hydrogel coated mesh compared to the control group; in these animals peritoneal healing was complete and predominantly by mesothelial cells with minimum fibrotic changes. Expression of inflammatory cytokines decreased compared to control animals, histology of abdominal organs, haematological and blood biochemical parameters remained normal. CONCLUSION: Therefore, silk hydrogel coating of polypropylene mesh can improve peritoneal healing, minimize adhesion formation, is safe and can augment the outcome of hernia surgery.

Processing silk hydrogel and its applications in biomedical materials.

This review mainly introduces the types of silk hydrogels, their processing methods, and applications. There are various methods for hydrogel preparation, and many new processes are being developed for various applications. Silk hydrogels can be used in cartilage tissue engineering, drug release materials, 3D scaffolds for cells, and artificial skin, among other applications because of their porous structure and high porosity and the large surface area for growth, migration, adhesion and proliferation of cells that the hydrogels provide. All of these advantages have made silk hydrogels increasingly attractive. In addition, silk hydrogels have wide prospects for application in the field of biomedical materials.