Superb Silk Hydrogels with High Adaptability, Bioactivity, and Versatility Enabled by Photo-Cross-Linking.

The exceptional biocompatibility and adaptability of hydrogels have garnered significant interest in the biomedical field for the fabrication of biomedical devices. However, conventional synthetic hydrogels still exhibit relatively weak and fragile properties. Drawing inspiration from the photosynthesis process, we developed a facile approach to achieve a harmonious combination of superior mechanical properties and efficient preparation of silk fibroin hydrogel through photo-cross-linking technology, accomplished within 60 s. The utilization of riboflavin and H2O2 enabled a sustainable cyclic photo-cross-linking reaction, facilitating the transformation from tyrosine to dityrosine and ultimately contributing to the formation of highly cross-linked hydrogels. These photo-cross-linking hydrogels exhibited excellent elasticity and restorability even after undergoing 1000 cycles of compression. Importantly, our findings presented that hydrogel-encapsulated adipose stem cells possess the ability to stimulate cell proliferation along with stem cell stemness. This was evidenced by the continuous high expression levels of OCT4 and SOX2 over 21 days. Additionally, the utilization of photo-cross-linking hydrogels can be extended to various material molding platforms, including microneedles, microcarriers, and bone screws. Consequently, this study offered a significant approach to fabricating biomedical hydrogels capable of facilitating real-time cell delivery, thereby introducing an innovative avenue for designing silk devices with exceptional machinability and adaptability in biomedical applications.

High biocompatible bone screw enabled by a rapid and robust chitosan/silk fibroin composite material.

Hydrogel has attracted tremendous attentions due to its excellent biocompatibility and adaptability in biomedical field. However, it is challenging by the conflicts between inadequate mechanical properties and service requirements. Herein, a rapid and robust hydrogel was developed by interpenetrating networks between chitosan and silk fibroin macromolecules. Thanks to these unique networks, the chitosan-based hydrogel exhibited superior mechanical performances. The maximum breaking strength, Young's modulus and swelling ratio of the hydrogel were 1187.8 kPa, 383.1 MPa and 4.5 % respectively. The hydrogel also supported the proliferation of human umbilical vein endothelial cells for 7 days. Notably, the hydrogel was easily molded into bone screw, and demonstrated compressive strengths of 45.7 MPa, Young's modulus of 675.6 MPa, respectively. After 49-day biodegradation, the residual rate of the screw in collagenase I solution was up to 89.6 % of the initial weight. In vitro, the screws not only had high resistance to biodegradation, but also had outstanding biocompatibility of osteoblast. This study provided a promising physical-chemical double crosslinking strategy to build orthopedic materials, holding a great potential in biomedical devices.

Fabrication of multifunctional silk nanofibril/hyaluronic acid scaffold for spinal cord repair.

Bioactive scaffolds accurately mimicking the structure and composition of the extracellular matrix have garnered significant interest in tissue engineering. In this study, we developed a platform utilizing natural silk nanofibrils, hyaluronic acid, and basic fibroblast growth factor for the purpose of promoting spinal cord regeneration by creating an optimal microenvironment. The bioactive scaffold exhibited notable characteristics such as high porosity and hydrophilicity, attributed to its unique nanostructure, high connectivity, and polysaccharide composition. Furthermore, the pore size of the scaffold can be adjusted within the range of 90 µm to 120 µm by varying the content of hyaluronic acid. In vitro, human umbilical vein endothelial cells were seeded into the scaffold, demonstrating enhanced cell viability. The scaffold facilitated cell proliferation and migration. In vivo experiments on rats indicated that the scaffold had a beneficial impact on spinal cord regeneration, creating a conducive environment for motor function recovery of the rats. This effect may be attributed to the scaffold's ability to stimulate axon growth and neuronal survival, as well as inhibit the formation of glial scars, as evidenced by the decreased expression of growth associated protein-43, microtubule-associated protein 2, and neurofilament-200. This study presents a promising method to develop a feasible bioscaffold for the treatment of spinal cord injury.

Development of a bioactive silk fibroin bilayer scaffold for wound healing and scar inhibition.

In cases of deep skin defects, spontaneous tissue regeneration and excessive collagen deposition lead to hyperplastic scars. Conventional remedial action after scar formation is limited with a high recurrence rate. In this study, we designed a new artificial skin bilayer using silk fibroin nanofibers films (SNF) as the epidermis, and silk fibroin (SF) / hyaluronic acid (HA) scaffold as the dermal layer. The regenerated SF film was used as a binder to form a functional SNF-SF-HA bilayer scaffold. The bilayer scaffold showed high porosity, hydrophilicity, and strength, and retained its shape over 30 days in PBS. In vitro, human umbilical vein endothelial cells were seeded into the bilayer scaffold and showed superior cell viability. In vivo analyses using the rabbit ear hypertrophic scar (HS) model indicated that the bilayer scaffold not only supported the reconstruction of new tissue, but also inhibited scar formation. The scaffold possibly achieved scar inhabitation by reducing wound contraction, weakening inflammatory reactions, and regulating collagen deposition and type conversion, which was partly observed through the downregulation of type I collagen, transforming growth factor-ß, and α-smooth muscle actin. This study describes a new strategy to expand the application of silk-based biomaterials for the treatment of hyperplastic skin scars.

Comparison of Silk Hydrogels Prepared via Different Methods.

Silk fibroin (SF) hydrogels have garnered extensive attention in biomedical materials, owing to their superior biological properties. However, the challenges facing the targeted silk fibroin hydrogels involve chemical agents and shortfalls in performance. In this study, the silk fibroin hydrogels were prepared in different ways: sonication induction, chemical crosslinking, photopolymerization, and enzyme-catalyzed crosslinking. The SF hydrogels derived from photopolymerization exhibited higher compressive properties, with 124 Kpa fracture compressive stress and breaks at about 46% compression. The chemical crosslinking and enzyme-catalyzed silk fibroin hydrogels showed superior toughness, yet sonication-induced hydrogels showed brittle performance resulting from an increase in silk II crystals. The chemical-crosslinked hydrogel demonstrated lower thermostability due to the weaker crosslinking degree. In vitro, all silk fibroin hydrogels supported the growth of human umbilical vein endothelial cells, as the cell viability of hydrogels without chemical agents was relatively higher. This study provides insights into the formation process of silk fibroin hydrogels and optimizes their design strategy for biomedical applications.

Controllable Production of Natural Silk Nanofibrils for Reinforcing Silk-Based Orthopedic Screws.

As a natural high-performance material with a unique hierarchical structure, silk is endowed with superior mechanical properties. However, the current approaches towards producing regenerated silk fibroin (SF) for the preparation of biomedical devices fail to fully exploit the mechanical potential of native silk materials. In this study, using a top-down approach, we exfoliated natural silk fibers into silk nanofibrils (SNFs), through the disintegration of interfibrillar binding forces. The as-prepared SNFs were employed to reinforce the regenerated SF solution to fabricate orthopedic screws with outstanding mechanical properties (compression modulus > 1.1 GPa in a hydrated state). Remarkably, these screws exhibited tunable biodegradation and high cytocompatibility. After 28 days of degradation in protease XIV solution, the weight loss of the screw was ~20% of the original weight. The screws offered a favorable microenvironment to human bone marrow mesenchymal stem cell growth and spread as determined by live/dead staining, F-action staining, and Alamar blue staining. The synergy between native structural components (SNFs) and regenerated SF solutions to form bionanocomposites provides a promising design strategy for the fabrication of biomedical devices with improved performance.

Natural silk nanofibers as building blocks for biomimetic aerogel scaffolds.

Protein nanofibers offer great promise for tissue engineering scaffolds owing to biomimetic architecture and exceptional biocompatibility. Natural silk nanofibrils (SNFs) are promising but unexplored protein nanofibers for biomedical applications. In this study, the SNF-assembled aerogel scaffolds with ECM-mimicking architecture and ultra-high porosity are developed based on a polysaccharides-assisted strategy. The SNFs exfoliated from silkworm silks can be utilized as building blocks to construct 3D nanofibrous scaffolds with tunable densities and desirable shapes on a large scale. We demonstrate that the natural polysaccharides can regulate SNF assembly through multiple binding modes, endowing the scaffolds with structural stability in water and tunable mechanical properties. As a proof of concept, the biocompatibility and biofunctionality of the chitosan-assembled SNF aerogels were investigated. The nanofibrous aerogels have excellent biocompatibility, and their biomimetic structure, ultra-high porosity, and large specific surface area endow the scaffolds with enhanced cell viability to mesenchymal stem cells. The nanofibrous aerogels were further functionalized by SNF-mediated biomineralization, demonstrating their potential as a bone-mimicking scaffold. Our results show the potential of natural nanostructured silks in the field of biomaterials and provide a feasible strategy to construct protein nanofiber scaffolds.

Recent progress in silk-based biosensors.

The applications of biosensors, in both medical and non-medical fields, have expanded enormously over the last few years. For conventional sensing applications, petrochemical materials, conducting polymers, metals and carbon-based materials are used. However, biosensing, especially in the medical field, places stringent material requirements of biocompatibility, biodegradability and other performance. Silk, an ancient natural organic material, is endowed with excellent biocompatibility, adjustable biodegradability, high strength and outstanding processability due to its unique physical and chemical structure. Owing to these exceptional properties, silk and its derivatives are of interest to researchers as biosensor matrices (supports). Herein, we review the representative research results of silk-based biosensors in medical and non-medical fields and highlight the applications and challenges of silk and its derivatives in medical biosensing applications. We envision that silk-based biosensors will become an integral part of the healthcare monitoring system in future.

Biomimetic Natural Silk Nanofibrous Microspheres for Multifunctional Biomedical Applications.

Silk nanofibrils (SNFs) extracted from natural silkworm silk represent a class of high-potential protein nanofiber material with unexplored biomedical applications. In this study, a SNF-assembled microsphere with extracellular matrix (ECM)-mimicking architecture and high specific surface area was developed. The SNFs were exfoliated from silkworm silks through an all-aqueous process and used as the building blocks for constructing the microspheres. Inspired by the structure and bioactive composition of ECM, hyaluronic acid (HA) was used as a bio-glue to regulate SNF assembly. With the assistance of HA, the SNF microspheres with stable fluffy nanofibrous structures were synthesized through electrospray. The biomimetic structure and nature derived composition endow the microspheres with excellent biocompatibility and enhanced osteogenic differentiation-inducing ability to mesenchymal stem cells. As proof of versatility, the SNF microspheres were further functionalized with other molecules and nanomaterials. Taking the advantages of the excellent blood compatibility and modifiability from the molecular level to the nanoscale of SNF microspheres, we demonstrated their versatile applications in protease detection and blood purification. On the basis of these results, we foresee that this natural silk-based nanofibrous microsphere may serve as a superior biomedical material for tissue engineering, early disease diagnosis, and therapeutic devices.

Advances in Skin Wound and Scar Repair by Polymer Scaffolds.

Scars, as the result of abnormal wound-healing response after skin injury, may lead to loss of aesthetics and physical dysfunction. Current clinical strategies, such as surgical excision, laser treatment, and drug application, provide late remedies for scarring, yet it is difficult to eliminate scars. In this review, the functions, roles of multiple polymer scaffolds in wound healing and scar inhibition are explored. Polysaccharide and protein scaffolds, an analog of extracellular matrix, act as templates for cell adhesion and migration, differentiation to facilitate wound reconstruction and limit scarring. Stem cell-seeded scaffolds and growth factors-loaded scaffolds offer significant bioactive substances to improve the wound healing process. Special emphasis is placed on scaffolds that continuously release oxygen, which greatly accelerates the vascularization process and ensures graft survival, providing convincing theoretical support and great promise for scarless healing.

Engineering vascularized dermal grafts by integrating a biomimetic scaffold and Wharton's jelly MSC-derived endothelial cells.

Tissue engineering aims to generate functional tissue constructs with the necessary scaffold properties for cell colonization and the establishment of a vascular network. However, treatment of tissue defects using synthetic scaffolds remains a challenge mainly due to insufficient and slow vascularization. Our previous study developed a macroporous silk fibroin scaffold with a nanofibrous microstructure, and demonstrated that the nanofibrous structure can promote the viability of endothelial cells (ECs) and guide cell migration. Further studies are needed to clarify the effect of scaffold microstructures on cell-mediated vascularization. Here, we investigated the efficacy of EC-seeded nanofibrous scaffolds in improving vascularization in vivo. ECs derived from induced human Wharton's Jelly mesenchymal stem cells served as a potential source for cell transplantation. The cell-seeded scaffolds were implanted into dermal defects of SD rats, demonstrating that the multiscale hierarchical design significantly improved the capacity of transplanted cells to promote and accelerate neovascularization and dermal reconstruction via enhancing cell infiltration, collagen deposition and growth factor expression. Our findings provide new insight into the development of degradable macroporous composite materials with 3D microstructures as tissue engineering scaffolds with enhanced vascularization functions, and also provide new treatment options for cell transplantation.

Natural Silk Nanofibril Aerogels with Distinctive Filtration Capacity and Heat-Retention Performance.

Nanofibrous aerogels have been extensively developed as multifunctional substrates in a wide range of fields. Natural silk nanofibrils (SNFs) are an appealing biopolymer due to their natural abundance, mechanical toughness, biodegradability, and excellent biocompatibility. However, fabricating 3D SNF materials with mechanical flexibility remains a challenge. Herein, SNF-based aerogels with controlled structures and well mechanical resilience were prepared. SNFs were extracted from silkworm silks by mechanical disintegration based on an all-aqueous system. The nanofibrils network and hierarchical cellular structure of the aerogels were tuned by the assembly of SNFs and foreign poly(vinyl alcohol) (PVA). The SNF aerogels exhibited an ultralow density (as low as 2.0 mg·cm-3) and well mechanical properties with a structure allowing for large deformations. These SNF aerogels demonstrated a reversible compression and stress retention after 100 cycles of compression. Furthermore, the resulting aerogels were used for air filtration and showed efficient filtration performance with a high dust-holding capacity and low resistance. Moreover, an extremely low thermal conductivity of 0.028 W·(m·K)-1 was achieved by the aerogel, showing its potential for use in heat-retention applications. This study provides a useful strategy for exploring the use of natural silks in 3D aerogels and offers options for developing filtration materials and ultralight heat-retention materials.

Natural silk nanofibrils as reinforcements for the preparation of chitosan-based bionanocomposites.

Nanofibrils derived from natural biopolymers have received extensive interest due to their exceptional mechanical properties and excellent biocompatibility. To fabricate biocompatible chitosan nanocomposites with high mechanical performance, silkworm silks were deconstructed into nanofibrils as structural and mechanical reinforcement of chitosan. After dispersing silk nanofibrils in chitosan solution, a set of nanocomposites, including film, porous scaffold, filament, and nanofibrous sponge, could be fabricated from the blended solutions. Silk nanofibrils could be uniformly dispersed in chitosan solution, and formed multi-dimensional nanocomposites. The nanocomposites exhibited enhanced mechanical strength and thermal stability, and provided a biomimetic nanofibrous structure for biomaterial applications. The enhancement in mechanical properties can be attributed to the interaction between the nanofibril phase and the chitosan matrix. As the polysaccharide/protein bionanocomposites derived from natural biopolymers, these materials offer new opportunities for biomaterial application by virtue of their biocompatibility and biodegradability, as well as enhanced mechanical properties and controllable mesoscopic structure.

Physically crosslinked silk fibroin/hyaluronic acid scaffolds.

Combining the properties of natural protein and polysaccharide is a promising strategy to generate bioactive biomaterials with controlled structure. Here, a new method of preparing water-insoluble silk fibroin/hyaluronic acid (SF/HA) scaffolds with tunable performances using an all-aqueous process is reported. Freezing-induced assembly was used to form silk I crystallization in the SF/HA blends. Silk I crystallization enhanced the stability of SF/HA scaffolds in water by forming silk I crystal networks to entrap blended HA without chemical cross-linking. Increasing HA content significantly enhanced the flexibility and water binding capacity of porous scaffolds, but high amount of HA reduced the water-stability of porous scaffolds due to insufficient silk I crystal cross-links. The enzymatic degradation behavior of the SF/HA scaffolds was investigated, revealing that the regulation ability of HA in the SF scaffolds. This novel nonchemically cross-linked protein/polysaccharide scaffold may be useful for soft tissue engineering due to excellent biocompatibility and tunable performances.

Topographic cues reveal filopodia-mediated cell locomotion in 3D microenvironment.

In cell-material interactions, the formation and functioning of filopodia have been demonstrated to be very sensitive to topographic cues. However, substrate-exploring functions of filopodia in a 3D microenvironment remain elusive. In this study, the silk fibroin film with a micropillar structure was prepared to reveal a filopodial-mediated cell response to 3D topographic cues. The micropillars provided a confined space for cell spreading by a simplified 3D structure, allowing initial cells to settle on the bottom of substrates rather than on the top of micropillars. Shortly after cell adhesion, the authors describe how cells transform from a filopodia-rich spherical cell state to a lamellipodia-dominated state that enables cell to climb along micropillars and spread on the top of the micropillars. The authors found that filopodia not only served as sensors for pathfinding but also provided nucleation scaffolds for the formation and orientation of minilamellipodia on the micropillar substrate. On the route of long filopodial extension following micropillars, all three functional filopodial adhesions have the ability to form veil-like minilamellipodium, simply by tethering the filopodium to the micropillars. Stable filopodia contacts consistently stimulated the local protrusion of a lamellipodium, which ultimately steered cell migration. Their results suggest the filopodia-mediated cell locomotion in the 3D microenvironment using a filopodia-to-minilamellipodium transformation mechanism.

Multichannel Bioactive Silk Nanofiber Conduits Direct and Enhance Axonal Regeneration after Spinal Cord Injury.

After a spinal cord injury, axonal regeneration over long distances is challenging due to the lack of physical guidance cues and bioactive signals. In this study, a multichannel bioactive silk fibroin nanofiber conduit was fabricated to improve spinal cord injury repair by enhancing axonal regeneration. The conduit was composed of longitudinally oriented silk fibroin nanofibers and then functionalized with laminin. In vitro, the bioactive conduits could promote neuron-like development and directional neurite extension of PC12 cells by providing a bioactive stimulus and physical guidance. In a spinal cord injury model in Sprague-Dawley rats, the biofunctionalized conduits displayed superior integration with the host tissue due to enhanced cell infiltration and tissue ingrowth. The glial scar was significantly reduced, allowing axonal ingrowth along with the channel direction. Compared to a single-channel conduit, the multichannel conduit improved spinal cord regeneration by boosting tissue ingrowth and axonal regeneration, indicating that the conduit architectures play critical roles in spinal cord regeneration. These silk fibroin conduits, along with the multichannel architecture, nanoscale cues, and the ability to bind bioactive compounds, represent promising candidates for spinal cord regeneration.

Biofunctionalized silk fibroin nanofibers for directional and long neurite outgrowth.

Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. Silk fibroin is a promising natural protein material for nerve repair. However, the lack of specific bioactive cues significantly hinders its application. In this study, the electrospun silk fibroin nanofibers with both biochemical and topographical cues were prepared. The alignment of electrospun nanofibers was optimized by controlling the surface linear velocity of a rotating drum. The silk fibroin nanofibers were further functionalized with laminin through covalent binding, confirmed by immunostaining observation. Cell proliferation and neurite outgrowth assays confirmed that the functionalized aligned nanofibers significantly enhanced directional axonal extensions, providing physical and bioactive cues for neurite outgrowth. Furthermore, the tubular scaffolds with longitudinally aligned microchannels were designed by rolling the functionalized silk fibroin nanofibers. The neurite extension across the lumen of the conduit along the direction of the aligned fibers is apparent. These results highlight the ability of laminin-immobilized silk fibroin nanofibers to enhance neurite outgrowth and to control directional neurite extension, providing a useful approach to construct a regenerative microenvironment for nerve repair materials.

Mechanically robust and flexible silk protein/polysaccharide composite sponges for wound dressing.

The cutaneous tissue contains cellular protein and polysaccharide components which together maintain the functionality of the tissue. In this study, silk fibroin (SF) and konjac glucomannan (KGM) were physically crosslinked to form biocompatible protein/polysaccharide sponges with tunable mechanical properties for wound dressing application. The pore structure of sponges can be adjusted by changing blend ratio of SF/KGM, forming homogeneous interconnected pore structure. FTIR and Raman results revealed the intermolecular interaction between SF and KGM, suggesting the formation of interpenetrating polymer network after ethanol/ammonium hydroxide treatment. Raising KGM content significantly enhanced water-absorption, water-retention abilities, and compression strength of porous sponges. Especially, the composite sponges possessed a similar compressive modulus with native skin tissue, showing a matched flexibility for wound treatment. Moreover, the cell viability results based on human dermal fibroblast cells demonstrated that the sponge showed excellent biocompatibility for cell adhesion and proliferation. Therefore, due to the strong water-absorption capacity, moist environment, similar compressive modulus with skin tissue and excellent biocompatibility, the composite sponges have potential application in wound dressings.

Comparative transcriptome analysis of salt-sensitive and salt-tolerant maize reveals potential mechanisms to enhance salt resistance.

BACKGROUND: Salt stress is a devastating environmental stress that causes plant growth inhibition and yield reduction. OBJECTIVE: The identification of salt-tolerant genes brings hope for the generation of salinity-tolerant crop plants through molecular breeding. METHODS: In this study, one salt-sensitive and one salt-tolerant maize inbred line were screened from 242 maize inbred lines. Reactive oxygen species (ROS)-related enzyme activities were detected and salt-responsive comparative transcriptome analysis was performed for control and 220 mM NaCl treated maize leaves. RESULTS: Salt-tolerant maize inbred line (L87) showed higher ROS-related enzyme (SOD, POD, APX and CAT) activities and accumulated relatively lower levels of ROS under salt stress. Of the total DEGs, 1856 upregulated DEGs were specific to L87, including stress tolerance-related members of the 70kDa family of heat shock proteins (Hsp70s) and aquaporins. The DEGs involved in the abscisic acid (ABA), ethylene, jasmonic acid (JA) and salicylic acid (SA) signal transduction pathways may determine the difference in salt tolerance between the two varieties, especially one central component SnRK2, that positively regulates ABA signaling and was only upregulated in L87. Analysis of DEGs related to ROS scavenging showed that some peroxidase (POD), glutathione S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) genes specific to L87 probably enhanced its salt tolerance. The analysis of differentially expressed transcription factors (TFs) suggested that WRKY TFs could contribute to the difference in salt tolerance between the two maize lines. CONCLUSION: Compared with Salt-sensitive maize inbred line (L29), L87 exhibits specific regulatory mechanisms related to salt tolerance, including plant hormone interactions, ROS scavenging and the regulation of TFs. Our study identifies new candidate genes that may regulate maize tolerance to salt stress and provides useful information for breeding maize with high salt resistance.

3D Printing of Silk Fibroin for Biomedical Applications.

Three-dimensional (3D) printing is regarded as a critical technological-evolution in material engineering, especially for customized biomedicine. However, a big challenge that hinders the 3D printing technique applied in biomedical field is applicable bioink. Silk fibroin (SF) is used as a biomaterial for decades due to its remarkable high machinability and good biocompatibility and biodegradability, which provides a possible alternate of bioink for 3D printing. In this review, we summarize the requirements, characteristics and processabilities of SF bioink, in particular, focusing on the printing possibilities and capabilities of bioink. Further, the current achievements of cell-loading SF based bioinks were comprehensively viewed from their physical properties, chemical components, and bioactivities as well. Finally, the emerging issues and prospects of SF based bioink for 3D printing are given. This review provides a reference for the programmable and multiple processes and the further improvement of silk-based biomaterials fabrication by 3D printing.