Subtle Regulation of Scaffold Stiffness for the Optimized Control of Cell Behavior

The rigidity of extracellular matrices can impact cell fate, guide tissue development, and initiate tumor formation. Scaffolds such as hydrogels with tunable levels of stiffness have been developed to control cell adhesion, migration, and differentiation, providing suitable microenvironments for different tissue outcomes. However, studies of cell–material interactions are largely confined to biomaterials with stiffness values that are coarsely regulated, so refinements in sensitive cellular responses and optimal stiffness values that determine cell fate remain elusive. Here, a freezing temperature, as a tunable regulating factor, was introduced to freeze-drying processes to form silk fibroin (SF) scaffolds with refined control of stiffness values. Due to this control of intermediate structural conformations of SF, the scaffolds exhibited differences in stiffness values to permit refined assessments of impact on cell behavior on cell-friendly surfaces. Both in vitro and in vivo results with these scaffolds exhibited gradually changeable cell migration and differentiation outcomes, as well as differences in tissue ingrowth, demonstrating the sensitivity of cellular responses to such refined mechanical cues. The optimal vascularization capacity of these SF scaffolds was in the 3–7.4 kPa range, suggesting a key range to develop bioactive biomaterials. Systematic fine regulation of scaffold rigidity based on the present strategy provides a platform for an improved understanding of cell–material interactions and also for generating optimized microenvironments for tissue regeneration

Engineered Tough Silk Hydrogels through Assembling β‑Sheet Rich Nanofibers Based on a Solvent Replacement Strategy

β-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 β-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

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 β-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

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

Silk–Hydroxyapatite Nanoscale Scaffolds with Programmable Growth Factor Delivery for Bone Repair

Osteoinductive biomaterials are attractive for repairing a variety of bone defects, and biomimetic strategies are useful toward developing bone scaffolds with such capacity. Here, a multiple biomimetic design was developed to improve the osteogenesis capacity of composite scaffolds consisting of hydroxyapatite nanoparticles (HA) and silk fibroin (SF). SF nanofibers and water-dispersible HA nanoparticles were blended to prepare the nanoscaled composite scaffolds with a uniform distribution of HA with a high HA content (40%), imitating the extracellular matrix (ECM) of bone. Bone morphogenetic protein-2 (BMP-2) was loaded in the SF scaffolds and HA to tune BMP-2 release. <i>In vitro</i> studies showed the preservation of BMP-2 bioactivity in the composite scaffolds, and programmable sustained release was achieved through adjusting the ratio of BMP-2 loaded on SF and HA. <i>In vitro</i> and <i>in vivo</i> osteogenesis studies demonstrated that the composite scaffolds showed improved osteogenesis capacity under suitable BMP-2 release conditions, significantly better than that of BMP-2 loaded SF–HA composite scaffolds reported previously. Therefore, these biomimetic SF–HA nanoscaled scaffolds with tunable BMP-2 delivery provide preferable microenvironments for bone regeneration

SilkGraphene Hybrid Hydrogels with Multiple Cues to Induce Nerve Cell Behavior

Cell behavior is dependent in part on chemical and physical cues from the extracellular matrix. Although the influence of various cues on cell behavior has been studied, challenges remain to incorporate multiple cues to matrix systems to optimize and control cell outcomes. Here, aligned silk fibroin (SF)-graphene hydrogels with preferable stiffness were developed through arranging SF nanofibers and SF-modified graphene sheets under an electric field. Different signals, such as bioactive graphene, nanofibrous structure, aligned topography, and mechanical stiffness, were tailored into the hydrogel system, providing niches for nerve cell responses. The desired adhesion, proliferation, differentiation, extensio,n and growth factor secretion of multiple nerve-related cells was achieved on these hydrogels, suggesting strong synergistic action through the combination of different cues. Based on the fabrication strategy, our present study provides a useful materials engineering platform for revealing cooperative influences of different signals on nerve cell behavior, to help in the understanding of cell–biomaterial interactions, with potential toward studies related to nerve regeneration

Tough Anisotropic Silk Nanofiber Hydrogels with Osteoinductive Capacity

Multiple physical cues such as hierarchical microstructures, topography, and stiffness influence cell fate during tissue regeneration. Yet, introducing multiple physical cues to the same biomaterial remains a challenge. Here, a synergistic cross-linking strategy was developed to fabricate protein hydrogels with multiple physical cues based on combinations of two types of silk nanofibers. β-sheet-rich silk nanofibers (BSNFs) were blended with amorphous silk nanofibers (ASNFs) to form composite nanofiber systems. The composites were transformed into tough hydrogels through horseradish peroxidase (HRP) cross-linking in an electric field, where ASNFs were cross-linked with HRP, while BSNFs were aligned by the electrical field. Anisotropic morphologies and higher stiffness of 120 kPa were achieved. These anisotropic hydrogels induced osteogenic differentiation and the aligned aggregation of stem cells in vitro while also exhibiting osteoinductive capacity in vivo. Improved tissue outcomes with the hydrogels suggest promising applications in bone tissue engineering, as the processing strategy described here provides options to form hydrogels with multiple physical cues

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

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

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