Simulation of Cortical and Cancellous Bone to Accelerate Tissue Regeneration.

Different tissues have complex anisotropic structures to support biological functions. Mimicking these complex structures in vitro remains a challenge in biomaterials designs in support of tissue regeneration. Here, inspired by different types of silk nanofibers, a composite materials strategy was pursued towards this challenge. A combination of fabrication methods was utilized to achieve separate control of amorphous and beta-sheet rich silk nanofibers in the same solution. Aqueous solutions containing these two structural types of silk nanofibers were then simultaneously treated with an electric field and with ethylene glycol diglycidyl ether (EGDE). Under these conditions, the beta-sheet rich silk nanofibers in the mixture responded to the electric field while the amorphous nanofibers were active in the crosslinking process with the EGDE. As a result, cryogels with anisotropic structures were prepared, including mimics for cortical- and cancellous-like bone biomaterials as a complex osteoinductive niche. In vitro studies revealed that mechanical cues of the cryogels induced osteodifferentiation of stem cells while the anisotropy inside the cryogels influenced immune reactions of macrophages. These bioactive cryogels also stimulated improved bone regeneration in vivo through modulation of inflammation, angiogenesis and osteogenesis responses, suggesting an effective strategy to develop bioactive matrices with complex anisotropic structures beneficial to tissue regeneration.

Macroporous Silk Nanofiber Cryogels with Tunable Properties.

Cryogels are widely used in tissue regeneration due to their porous structures and friendly hydrogel performance. Silk-based cryogels were developed but failed to exhibit desirable tunable properties to adapt various biomedical applications. Here, amorphous short silk nanofibers (SSFs) were introduced to fabricate silk cryogels with versatile cues. Compared to previous silk cryogels, the SSF cryogels prepared under same conditions showed significantly enhanced mechanical properties. The microporous cryogels were achieved under lower silk concentrations, confirming better tunability. Versatile cryogels with the modulus in the range of 0.5-283.7 kPa were developed through adjusting silk concentration and crosslinking conditions, superior to previous silk cryogel systems. Besides better cytocompatibility, the SSF cryogels were endowed with effective mechanical cues to control osteogenetic differentiation behaviors of BMSCs. The mechanical properties could be further regulated finely through the introduction of ß-sheet-rich silk nanofibers (SNFs), which suggested possible optimization of mechanical niches. Bioactive cargo-laden SNFs were introduced to the SSF cryogel systems, bringing biochemical signals without the compromise of mechanical properties. Versatile SNF-based cryogels with different physical and biological cues were developed here to facilitate the applications in various tissue engineering.

Natural Nanofiber Shuttles for Transporting Hydrophobic Cargo into Aqueous Solutions.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Fabrication of Silk Scaffolds with Nanomicroscaled Structures and Tunable Stiffness.

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce ß-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced ß-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk.

Nanosized Silk-Magnesium Complexes for Tissue Regeneration.

Metal ions provide multifunctional signals for cell and tissue functions, including regeneration. Inspired by metal-organic frameworks (MOFs), nanosized silk protein aggregates with a high negative charge density are used to form stable silk-magnesium ion complexes. Magnesium ions (Mg ions) are added directly to silk nanoparticle solutions, inducing gelation through the formation of silk-Mg coordination complexes. The Mg ions are released slowly from the nanoparticles through diffusion, with sustained release via tuning the degradation or dissolution of the nanosized silk aggregates. Studies in vitro reveal a dose-dependent influence of Mg ions on angiogenic and anti-inflammatory functions. Silk-Mg ion complexes in the form of hydrogels also stimulate tissue regeneration with a reduced formation of scar tissue in vivo, suggesting potential utility in tissue regeneration.

Tough Porous Silk Nanofiber-Derived Cryogels with Osteogenic and Angiogenic Capacity for Bone Repair.

Tough porous cryogels with angiogenesis and osteogenesis features remain a design challenge for utility in bone regeneration. Here, building off of the recent efforts to generate tough silk nanofiber-derived cryogels with osteogenic activity, deferoxamine (DFO) is loaded in silk nanofiber-derived cryogels to introduce angiogenic capacity. Both the mechanical cues (stiffness) and the sustained release of DFO from the gels are controlled by tuning the concentration of silk nanofibers in the system, achieving a modulus above 400 kPa and slow release of the DFO over 60 days. The modulus of the cryogels and the released DFO induce osteogenic and angiogenic activity, which facilitates bone regeneration in vivo in femur defects in rat, resulting in faster regeneration of vascularized bone tissue. The tunable physical and chemical cues derived from these nanofibrous-microporous structures support the potential for silk cryogels in bone tissue regeneration.

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.

Anisotropic silk nanofiber layers as regulators of angiogenesis for optimized bone regeneration.

Osteogenesis-angiogenesis coupling processes play a crucial role in bone regeneration. Here, electric field induced aligned nanofiber layers with tunable thickness were coated on the surface of pore walls inside the deferoxamine (DFO)-laden silk fibroin (SF) and hydroxyapatite (HA) composite scaffolds to regulate the release of DFO to control vascularization dynamically. Longer electric field treatments resulted in gradually thickening layers to reduce the release rate of DFO where the released amount of DFO decreased gradually from 84% to 63% after 28 days. Besides the osteogenic capacity of HA, the changeable release of DFO brought different angiogenic behaviors in bone regeneration process, which provided a desirable niche with osteogenic and angiogenic cues. Anisotropic cues were introduced to facilitate cell migration inside the scaffolds. Changeable cytokine secretion from endothelial cells cultured in the different scaffolds revealed the regulation of cell responses related to vascularization in vitro. Peak expression of angiogenic factors appeared at days 7, 21 and 35 for endothelial cells cultured in the scaffolds with different silk nanofier layers, suggesting the dynamical regulation of angiogenesis. Although all of the scaffolds had the same silk and HA composition, in vitro cell studies indicated different osteogenic capacities for the scaffolds, suggesting that the regulation of DFO release also influenced osteogenesis outcomes in vitro. In vivo, the best bone regeneration occurred in defects treated with the composite scaffolds that exhibited the best osteogenic capacity in vitro. Using a rat bone defect model, healing was achieved within 12 weeks, superior to those treated with previous SF-HA composite matrices. Controlling angiogenic properties of bone biomaterials dynamically is an effective strategy to improve bone regeneration capacity.

Silk Nanocarrier Size Optimization for Enhanced Tumor Cell Penetration and Cytotoxicity In Vitro.

Silk nanofibers are versatile carriers for hydrophobic and hydrophilic drugs, but fall short in terms of effective delivery to cells, which is essential for therapeutic benefits. Here, the size of silk nanofibers was tuned by ultrasonic treatment to improve the cell penetration features without impacting the structural features. The gradual decrease in silk nanofiber length from 1700 to 40 nm resulted in improved cell uptake. The internalized silk nanofiber carriers evaded lysosomes, which facilitated retention in cancer cells in vitro. The smaller sizes also facilitated enhanced penetration of tumor spheroids for improved delivery in vitro. The cytotoxicity of paclitaxel (PTX)-laden nanocarriers increased when the length of the silk nanocarriers decreased. Both the drug loading capacity and delivery of silk nanocarriers with optimized sizes suggest potential utility in cell treatments.

Nerve Growth Factor-Laden Anisotropic Silk Nanofiber Hydrogels to Regulate Neuronal/Astroglial Differentiation for Scarless Spinal Cord Repair.

Scarless spinal cord regeneration remains a challenge due to the complicated microenvironment at lesion sites. In this study, the nerve growth factor (NGF) was immobilized in silk protein nanofiber hydrogels with hierarchical anisotropic microstructures to fabricate bioactive systems that provide multiple physical and biological cues to address spinal cord injury (SCI). The NGF maintained bioactivity inside the hydrogels and regulated the neuronal/astroglial differentiation of neural stem cells. The aligned microstructures facilitated the migration and orientation of cells, which further stimulated angiogenesis and neuron extensions both in vitro and in vivo. In a severe rat long-span hemisection SCI model, these hydrogel matrices reduced scar formation and achieved the scarless repair of the spinal cord and effective recovery of motor functions. Histological analysis confirmed the directional regenerated neuronal tissues, with a similar morphology to that of the normal spinal cord. The in vitro and in vivo results showed promising utility for these NGF-laden silk hydrogels for spinal cord regeneration while also demonstrating the feasibility of cell-free bioactive matrices with multiple cues to regulate endogenous cell responses.

Silk Biomaterials for Bone Tissue Engineering.

Silk is a natural fibrous polymer with application potential in regenerative medicine. Increasing interest remains for silk materials in bone tissue engineering due to their characteristics in biocompatibility, biodegradability and mechanical properties. Plenty of the in vitro and in vivo studies confirmed the advantages of silk in accelerating bone regeneration. Silk is processed into scaffolds, hydrogels, and films to facilitate different bone regenerative applications. Bioactive factors such as growth factors and drugs, and stem cells are introduced to silk-based matrices to create friendly and osteogenic microenvironments, directing cell behaviors and bone regeneration. The recent progress in silk-based bone biomaterials is discussed and focused on different fabrication and functionalization methods related to osteogenesis. The challenges and potential targets of silk bone materials are highlighted to evaluate the future development of silk-based bone materials.

Fragile-Tough Mechanical Reversion of Silk Materials via Tuning Supramolecular Assembly.

Regenerated silk nanofibers are interesting as protein-based material building blocks due to their unique structure and biological origin. Here, a new strategy based on control of supramolecular assembly was developed to regulate interactions among silk nanofibers by changing the solvent, achieving tough mechanical features for silk films. Formic acid was used to replace water related to charge repulsion of silk nanofibers in solution, inducing interactions among the nanofibers. The films formed under these conditions had an elastic modulus of 3.4 ± 0.3 GPa, an ultimate tensile strength of 76.9 ± 1.6 MPa, and an elongation at break of 3.5 ± 0.1%, while the materials formed from aqueous solutions remained fragile. The mechanical performance of the formic acid-derived nanofiber films was further improved through post-stretching or via the addition of graphene. In addition, the silk nanofiber films could be functionalized with various bioactive ingredients such as curcumin. These new silk nanofiber films with a unique combination of mechanical properties and functions provide new biomaterials achieved using traditional solvents and processes through insight and control of their assembly mechanisms in solution.

Injectable silk nanofiber hydrogels as stem cell carriers to accelerate wound healing.

Stem cells have potential utility in wound therapy, however the benefits are often limited due to cell injury from shear stress during injection and poor retention at the wound site. Here, shear-thinning silk nanofiber hydrogels were used to load bone marrow derived mesenchymal stem cells (BMSCs) and inject into wound sites to optimize cell retention and accelerate wound healing. The BMSCs in the silk nanofiber hydrogels maintained stemness better than the cells cultured on plates, and the expression of wound healing-related genes was significantly higher in the hydrogels with higher silk concentrations (2 wt%). The silk nanofibers physically prevented migration of BMSCs from the deposition site in the wound bed. In addition to faster wound healing, these BMSC-loaded hydrogels mediated angiogenesis and inflammation and improved collagen deposition and hair follicle regeneration in vivo in rats. Considering that these silk nanofiber hydrogels were successfully used here as carriers for stem cells to accelerate wound healing, further study for skin regeneration may be warranted.

Asiaticoside-laden silk nanofiber hydrogels to regulate inflammation and angiogenesis for scarless skin regeneration.

Scarless skin regeneration remains a challenge due to the complicated microenvironment involved in wound healing. Here, the hydrophobic drug, asiaticoside (AC), was loaded inside silk nanofiber hydrogels to achieve bioactive and injectable matrices for skin regeneration. AC was dispersed in aqueous silk nanofiber hydrogels with retention of biological functions that regulated inflammatory reactions and vascularization in vitro. After implantation in full-thickness wound defects, these AC-laden hydrogel matrices achieved scarless wound repair. Inflammatory reactions and angiogenesis were regulated during inflammation and remodeling, which was responsible for wound regeneration similar to normal skin. Both in vitro and in vivo studies demonstrated promising applications of these AC-laden silk hydrogels towards scarless tissue regeneration.

Pressure-driven spreadable deferoxamine-laden hydrogels for vascularized skin flaps.

The development of hydrogels that support vascularization to improve the survival of skin flaps, yet establishing homogeneous angiogenic niches without compromising the ease of use in surgical settings remains a challenge. Here, pressure-driven spreadable hydrogels were developed utilizing beta-sheet rich silk nanofiber materials. These silk nanofiber-based hydrogels exhibited excellent spreading under mild pressure to form a thin coating to cover all the regions of the skin flaps. Deferoxamine (DFO) was loaded onto the silk nanofibers to support vascularization and these DFO-laden hydrogels were implanted under skin flaps in rats to fill the interface between the wound bed and the flap using the applied pressure. The thickness of the spread hydrogels was below 200 µm, minimizing the physical barrier effects from the hydrogels. The distribution of the hydrogels provided homogeneous angiogenic stimulation, accelerating rapid blood vessel network formation and significantly improving the survival of the skin flaps. The hydrogels also modulated the immune reactions, further facilitating the regeneration of the skin flaps. Considering the homogeneous distribution at the wound sites, improved vascularization, reduced barrier effects and low inflammation, these hydrogels appear to be promising candidates for use in tissue repair where a high blood supply is in demand. The pressure-driven spreading properties should simplify the use of the hydrogels in surgical settings to facilitate clinical translation.

Nerve Guidance Conduits with Hierarchical Anisotropic Architecture for Peripheral Nerve Regeneration.

Nerve guidance conduits with multifunctional features could offer microenvironments for improved nerve regeneration and functional recovery. However, the challenge remains to optimize multiple cues in nerve conduit systems due to the interplay of these factors during fabrication. Here, a modular assembly for the fabrication of nerve conduits is utilized to address the goal of incorporating multifunctional guidance cues for nerve regeneration. Silk-based hollow conduits with suitable size and mechanical properties, along with silk nanofiber fillers with tunable hierarchical anisotropic architectures and microporous structures, are developed and assembled into conduits. These conduits supported improves nerve regeneration in terms of cell proliferation (Schwann and PC12 cells) and growth factor secretion (BDNF, brain-derived neurotrophic factor) in vitro, and the in vivo repair and functional recovery of rat sciatic nerve defects. Nerve regeneration using these new conduit designs is comparable to autografts, providing a path towards future clinical impact.

Injectable Desferrioxamine-Laden Silk Nanofiber Hydrogels for Accelerating Diabetic Wound Healing.

Dysangiogenesis and chronic inflammation are two critical reasons for diabetic foot ulcers. Desferrioxamine (DFO) was used clinically in the treatment of diabetic foot ulcers by repeated injections because of its capacity to induce vascularization. Biocompatible carriers that release DFO slowly and facilitate healing simultaneously are preferable options to accelerate the healing of diabetic wounds. Here, DFO-laden silk nanofiber hydrogels that provided a sustained release of DFO for more than 40 days were used to treat diabetic wounds. The DFO-laden hydrogels stimulated the healing of diabetic wounds. In vitro cell studies revealed that the DFO-laden hydrogels modulated the migration and gene expression of endothelial cells, and they also tuned the inflammation behavior of macrophages. These results were confirmed in an in vivo diabetic wound model. The DFO-laden hydrogels alleviated dysangiogenesis and chronic inflammation in the diabetic wounds, resulting in a more rapid wound healing and increased collagen deposition. Both in vitro and in vivo studies suggested potential clinical applications of these DFO-laden hydrogels in the treatment of diabetic ulcers.

Blastocyst-Inspired Hydrogels to Maintain Undifferentiation of Mouse Embryonic Stem Cells.

Stem cell fate is determined by specific niches that provide multiple physical, chemical, and biological cues. However, the hierarchy or cascade of impact of these cues remains elusive due to their spatiotemporal complexity. Here, anisotropic silk protein nanofiber-based hydrogels with suitable cell adhesion capacity are developed to mimic the physical microenvironment inside the blastocele. The hydrogels enable mouse embryonic stem cells (mESCs) to maintain stemness in vitro in the absence of both leukemia inhibitory factor (LIF) and mouse embryonic fibroblasts (MEFs), two critical factors in the standard protocol for mESC maintenance. The mESCs on hydrogels can achieve superior pluripotency, genetic stability, developmental capacity, and germline transmission to those cultured with the standard protocol. Such biomaterials establish an improved dynamic niche through stimulating the secretion of autocrine factors and are sufficient to maintain the pluripotency and propagation of ESCs. The mESCs on hydrogels are distinct in their expression profiles and more resemble ESCs in vivo. The physical cues can thus initiate a self-sustaining stemness-maintaining program. In addition to providing a relatively simple and low-cost option for expansion and utility of ESCs in biological research and therapeutic applications, this biomimetic material helps gain more insights into the underpinnings of early mammalian embryogenesis.

Water-insoluble amorphous silk fibroin scaffolds from aqueous solutions.

Regenerated silk fibroin (RSF) is emerging as promising biomaterial for regeneration, drug delivery and optical devices, with continued demand for mild, all-aqueous processes to control microstructure and the performance. Here, temperature control of assembly kinetics was introduced to prepare the water-insoluble scaffolds from neutral aqueous solutions of RSF protein. Higher temperatures were used to accelerate the assembly rate of the silk fibroin protein chains in aqueous solution and during the lyophilization process, resulting in water-insoluble scaffold formation. The scaffolds were mainly composed of amorphous states of the silk fibroin chains, endowing softer mechanical properties. These scaffolds also showed nanofibrous structures, improved cell proliferation in vitro and enhanced neovascularization and tissue regeneration in vivo than previously reported silk fibroin scaffolds. These results suggest utility of silk scaffolds in soft tissue regeneration.

Microfluidic Silk Fibers with Aligned Hierarchical Microstructures.

The hierarchical structure of the ECM provides specific niches for tissues to regulate cell behavior, yet the challenge remains to design biomaterial systems for tissue regeneration to recreate such features in vitro. Here, we achieved this goal through the use of aligned hierarchical structures of native silk fibers, generated through the integration of "bottom-up" and "top-down" strategies to generate regenerated silk fibers with aligned nano- to micro-hierarchical structures. To achieve these designs, we assembled and dispersed silk nanofibers (SNF) in formic acid and spun them into fibers using bioinspired microfluidic chips with a geometry mimicking the native silk gland. The fibers generated using this device exhibited aligned hierarchical structure with fiber mechanical properties superior to fibers derived from more traditional spinning approaches with regenerated silk solutions. Besides the improved mechanical properties, Raman spectroscopic results indicated similarly aligned structures to native fibers and active control of cell proliferation, migration, and aggregate orientation. The results indicate the feasibility of developing bioactive silk fiber materials with hierarchical structures to facilitate utility in a range of cell and tissue regeneration scenarios.