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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.

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.

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.

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.

Bone regeneration is a complex process in which angiogenesis and osteogenesis are crucial. Introducing multiple angiogenic and osteogenic cues simultaneously into a single system and tuning these cues to optimize the niche remains a challenge for bone tissue engineering. Herein, based on our injectable biomimetic hydrogels composed of silk nanofibers (SNF) and hydroxyapatite nanoparticles (HA), deferoxamine (DFO) and bone morphogenetic protein-2 (BMP-2) were loaded on SNF and HA to introduce more angiogenic and osteogenic cues. The angiogenesis and osteogenesis capacity of injectable hydrogels could be regulated by tuning the delivery of DFO and BMP-2 independently, resulting in vascularization and bone regeneration in cranial defects. The angiogenesis and osteogenesis outcomes accelerated the regeneration of vascularized bones toward similar composition and structure to natural bones. Therefore, the multiple biophysical and chemical cues provided by the nanofibrous structures, organic-inorganic compositions, and chemical and biochemical angiogenic and osteogenic inducing cues suggest the potential for clinical applicability of these hydrogels in bone tissue engineering.

Scarless skin regeneration with functional tissue remains a challenge for full-thickness wounds. Here, mesenchymal stem cell (MSC)-laden hydrogels are developed for scarless wound healing with hair follicles. Microgels composed of aligned silk nanofibers are used to load MSCs to modulate the paracrine. MSC-laden microgels are dispersed into injectable silk nanofiber hydrogels, forming composites biomaterials containing the cells. The injectable hydrogels protect and stabilize the MSCs in the wounds. The synergistic action of silk-based composite hydrogels and MSCs stimulated angiogenesis and M1-M2 phenotype switching of macrophages, provides a suitable niche for functional recovery of wounds. Compared to skin defects treated with MSC-free hydrogels, the defects treated with the MSC-laden composite hydrogels heal faster and form scarless tissues with hair follicles. Wound healing can be further improved by adjusting the ratio of silk nanofibers and particles and the loaded MSCs, suggesting tunability of the system. To the best of current knowledge, this is the first time scarless skin regeneration with hair follicles based on silk material systems is reported. The improved wound healing capacity of the systems suggests future in vivo studies to compare to other biomaterial systems related to clinical goals in skin regeneration in the absence of scarring., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Gradient biomaterials are considered as preferable matrices for tissue engineering due to better simulation of native tissues. The introduction of gradient cues usually needs special equipment and complex process but is only effective to limited biomaterials. Incorporation of multiple gradients in the hydrogels remains challenges. Here, beta-sheet rich silk nanofibers (BSNF) were used as building blocks to introduce multiple gradients into different hydrogel systems through the joint action of crosslinking and electric field. The blocks migrated to the anode along the electric field and gradually stagnated due to the solution-hydrogel transition of the systems, finally achieving gradient distribution of the blocks in the formed hydrogels. The gradient distribution of the blocks could be tuned easily through changing different factors such as solution viscosity, which resulted in highly tunable gradient of mechanical cues. The blocks were also aligned under the electric field, endowing orientation gradient simultaneously. Different cargos could be loaded on the blocks and form gradient cues through the same crosslinking-electric field strategy. The building blocks could be introduced to various hydrogels such as Gelatin and NIPAM, indicating the universality. Complex niches with multiple gradient cues could be achieved through the strategy. Silk-based hydrogels with suitable mechanical gradients were fabricated to control the osteogenesis and chondrogenesis. Chondrogenic-osteogenic gradient transition was obtained, which stimulated the ectopic osteochondral tissue regeneration in vivo. The versatility and highly controllability of the strategy as well as multifunction of the building blocks reveal the applicability in complex tissue engineering and various interfacial tissues.

Silk fibroin (SF) hydrogels have been engineered as universal substrates for various tissue regenerations and drug delivery. Although different physical and chemical crosslinking strategies are developed to form SF hydrogels with suitable performances, a significant gap remains to match specific requirements of various tissues. Here, amorphous SF nanofibers with more tyrosine residues outside the surfaces are used to replace traditional SF. Under the same crosslinking conditions, the use of amorphous SF nanofibers results in tougher properties, four times higher stiffness than that from traditional SF solutions. Unlike previous SF hydrogels, the SF nanofiber hydrogels show high tunability in wide modulus range of 0.6-160 kPa under low SF concentrations (below 5 wt%), showing improved mechanical match with various soft tissues. Better stability and cytocompatibility are also achieved, further confirming the superiority of the hydrogels as the tissue substrates. Therefore, a feasible strategy is developed to optimize the performances of SF hydrogel via tuning the nano-structural state in aqueous solutions, which will enrich SF-based hydrogel family in future., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The article presents a study of hydrogel assembly with hierarchical alignment of material or anisotropy to balance electrostatic forces. Topics discussed include the charged silk nanofibers aligns due to the electrostatic interactions between charged silk nanofibers and the electric field, the unexplained several phenomena in the gelatin process such as the separation of the layers and homogenous distance between layers inside and hydrogels.