Silk-protein-based gradient hydrogels with multimode reprogrammable shape changes for biointegrated devices.

Biocompatible and morphable hydrogels capable of multimode reprogrammable, and adaptive shape changes are potentially useful for diverse biomedical applications. However, existing morphable systems often rely on complicated structural designs involving cumbersome and energy-intensive fabrication processes. Here, we report a simple electric-field-activated protein network migration strategy to reversibly program silk-protein hydrogels with controllable and reprogrammable complex shape transformations. The application of a low electric field enables the convergence of net negatively charged protein cross-linking networks toward the anode (isoelectric point plane) due to the pH gradient generated in the process, facilitating the formation of a gradient network structure and systems suitable for three-dimensional shape change. These tunable protein networks can be reprogrammed or permanently fixed by control of the polymorphic transitions. We show that these morphing hydrogels are capable of conformally interfacing with biological tissues by programming the shape changes and a bimorph structure consisting of aligned carbon nanotube multilayers and the silk hydrogels was assembled to illustrate utility as an implantable bioelectronic device for localized low-voltage electrical stimulation of the sciatic nerve in a rabbit.

Development of ovalbumin implants with different spatial configurations for treatment of peripheral nerve injury.

Peripheral nerve injury (PNI) seriously affects the health and life of patients, and is an urgent clinical problem that needs to be resolved. Nerve implants prepared from various biomaterials have played a positive role in PNI, but the effect should be further improved and thus new biomaterials is urgently needed. Ovalbumin (OVA) contains a variety of bioactive components, low immunogenicity, tolerance, antimicrobial activity, non-toxicity and biodegradability, and has the ability to promote wound healing, cell growth and antimicrobial properties. However, there are few studies on the application of OVA in neural tissue engineering. In this study, OVA implants with different spatial structures (membrane, fiber, and lyophilized scaffolds) were constructed by casting, electrospinning, and freeze-drying methods, respectively. The results showed that the OVA implants had excellent physicochemical properties and were biocompatible without significant toxicity, and can promote vascularization, show good histocompatibility, without excessive inflammatory response and immunogenicity. The in vitro results showed that OVA implants could promote the proliferation and migration of Schwann cells, while the in vivo results confirmed that OVA implants (the E5/70% and 20 kV 20 µL/min groups) could effectively regulate the growth of blood vessels, reduce the inflammatory response and promote the repair of subcutaneous nerve injury. Further on, the high-throughput sequencing results showed that the OVA implants up-regulated differential expression of genes related to biological processes such as tumor necrosis factor-α (TNF-α), phosphatidylinositide 3-kinases/protein kinase B (PI3K-Akt) signaling pathway, axon guidance, cellular adhesion junctions, and nerve regeneration in Schwann cells. The present study is expected to provide new design concepts and theoretical accumulation for the development of a new generation of nerve regeneration implantable biomaterials.

Biomimetic-inspired piezoelectric ovalbumin/BaTiO3 scaffolds synergizing with anisotropic topology for modulating Schwann cell and DRG behavior.

The treatment of peripheral nerve injury is a clinical challenge that tremendously affected the patients' health and life. Anisotropic topographies and electric cues can simulate the regenerative microenvironment of nerve from physical and biological aspects, which show promising application in nerve regeneration. However, most studies just unilaterally emphasize the effect of sole topological- or electric- cue on nerve regeneration, while rarely considering the synergistic function of both cues simultaneously. In this study, a biomimetic-inspired piezoelectric topological ovalbumin/BaTiO3 scaffold that can provide non-invasive electrical stimulation in situ was constructed by combining piezoelectric BaTiO3 nanoparticles and surface microtopography. The results showed that the incorporation of piezoelectric nanoparticles could improve the mechanical properties of the scaffolds, and the piezoelectric output of the scaffolds after polarization was significantly increased. Biological evaluation revealed that the piezoelectric topological scaffolds could regulate the orientation growth of SCs, promote axon elongation of DRG, and upregulate the genes expression referring to myelination and axon growth, thus rapidly integrated chemical-mechanical signals and transmitted them for effectively promoting neuronal myelination, which was closely related to peripheral neurogenesis. The study suggests that the anisotropic surface topology combined with non-invasive electronic stimulation of the ovalbumin/BaTiO3 scaffolds possess a promising application prospect in the repair and regeneration of peripheral nerve injury.

IKVAV functionalized oriented PCL/Fe3O4 scaffolds for magnetically modulating DRG growth behavior.

The re-bridging of the deficient nerve is the main problem to be solved after the functional impairment of the peripheral nerve. In this study, a directionally aligned polycaprolactone/triiron tetraoxide (PCL/Fe3O4) fiber scaffolds were firstly prepared by electrospinning technique, and further then grafted with IKVAV peptide for regulating DRG growth and axon extension in peripheral nerve regeneration. The results showed that oriented aligned magnetic PCL/Fe3O4 composite scaffolds were successfully prepared by electrospinning technique and possessed good mechanical properties and magnetic responsiveness. The PCL/Fe3O4 scaffolds containing different Fe3O4 concentrations were free of cytotoxicity, indicating the good biocompatibility and low cytotoxicity of the scaffolds. The IKVAV-functionalized PCL/Fe3O4 scaffolds were able to guide and promote the directional extension of axons, the application of external magnetic field and the grafting of IKVAV peptides significantly further promoted the growth of DRGs and axons. The ELISA test results showed that the AP-10 F group scaffolds promoted the secretion of nerve growth factor (NGF) from DRG under a static magnetic field (SMF), thus promoting the growth and extension of axons. Importantly, the IKVAV-functionalized PCL/Fe3O4 scaffolds could significantly up-regulate the expression of Cntn2, PCNA, Sox10 and Isca1 genes related to adhesion, proliferation and magnetic receptor function under the stimulation of SMF. Therefore, IKVAV-functionalized PCL/Fe3O4 composite oriented scaffolds have potential applications in neural tissue engineering.

Multi-scale, multi-level anisotropic silk fibroin/metformin scaffolds for repair of peripheral nerve injury.

Silk fibroin (SF) as a natural polymer has a long history of application in various regenerative medicine fields, but there are still many shortcomings in silk fibroin for using as nerve scaffolds, which limit its clinical application in peripheral nerve regeneration (PNR). In this work, a multi-scale and multi-level metformin (MF)-loaded silk fibroin scaffold with anisotropic micro-nano composite topology was prepared by micromolding electrospinning for accelerating PNR. The scaffolds were characterized for morphology, wettability, mechanical properties, degradability, and drug release, and Schwann cells (SCs) and dorsal root ganglia (DRG) were cultured on the scaffolds to assess their effects on neural cell behavior. Finally, the gene expression differences of neural cells cultured on scaffolds were analyzed by gene sequencing and RT-qPCR to explore the possible signaling pathways and mechanisms. The results showed that the scaffolds had excellent mechanical properties and hydrophilicity, slow degradation rate and drug release rate, which were enough to support the repair of peripheral nerve injury for a long time. In Vitro cell experiments showed that the scaffolds could significantly promote the orientation of SCs and axons extension of DRG. Gene sequencing and RT-qPCR revealed that the scaffolds could up-regulate the expression of genes related to SCs proliferation, adhesion, migration, and myelination. In summary, the scaffolds hold great potential for promoting PNR at the micro/nano multiscale and physical/chemical levels and show promising application for the treatment of peripheral nerve injury in the future.

Surface topologized ovalbumin scaffolds containing YIGSR peptides for modulating Schwann cell behavior.

Peripheral nerve injuries (PNI) currently have limited therapeutic efficacy, and functional scaffolds have been shown to be effective for treating PNI. Ovalbumin (OVA) is widely used as a natural biomaterial for repairing damaged tissues due to its excellent biocompatibility and the presence of various bioactive components. However, there are few reports on the repair of PNI by ovalbumin. In this study, a novel bionic functionalized topological scaffold based on ovalbumin and grafted with tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide was constructed by micro-molding method and surface-biomodification technology. The scaffolds were subjected to a series of evaluations in terms of morphology, mechanics, hydrophilicity, and biocompatibility, and the related molecular mechanisms were further penetrated. The results showed that the scaffolds prepared in this study had aligned ridge/groove structure, good mechanical properties and biocompatibility, and could be used as carriers to slowly release YIGSR, which effectively promoted the proliferation, migration and elongation of Schwann Cells (SCs), and significantly up-regulated the gene expression related to proliferation, apoptosis, migration and axon regeneration. Therefore, the bionic functional topological scaffold has significant application potential for promoting peripheral nerve regeneration and provides a new therapeutic option for repairing PNI.

Piezoelectric materials for neuroregeneration: a review.

The purpose of nerve regeneration via tissue engineering strategies is to create a microenvironment that mimics natural nerve growth for achieving functional recovery. Biomaterial scaffolds offer a promising option for the clinical treatment of large nerve gaps due to the rapid advancement of materials science and regenerative medicine. The design of biomimetic scaffolds should take into account the inherent properties of the nerve and its growth environment, such as stiffness, topography, adhesion, conductivity, and chemical functionality. Various advanced techniques have been employed to develop suitable scaffolds for nerve repair. Since neuronal cells have electrical activity, the transmission of bioelectrical signals is crucial for the functional recovery of nerves. Therefore, an ideal peripheral nerve scaffold should have electrical activity properties similar to those of natural nerves, in addition to a delicate structure. Piezoelectric materials can convert stress changes into electrical signals that can activate different intracellular signaling pathways critical for cell activity and function, which makes them potentially useful for nerve tissue regeneration. However, a comprehensive review of piezoelectric materials for neuroregeneration is still lacking. Thus, this review systematically summarizes the development of piezoelectric materials and their application in the field of nerve regeneration. First, the electrical signals and natural piezoelectricity phenomenon in various organisms are briefly introduced. Second, the most commonly used piezoelectric materials in neural tissue engineering, including biocompatible piezoelectric polymers, inorganic piezoelectric materials, and natural piezoelectric materials, are classified and discussed. Finally, the challenges and future research directions of piezoelectric materials for application in nerve regeneration are proposed.

Electrospinning porcine decellularized nerve matrix scaffold for peripheral nerve regeneration.

The composition and spatial structure of bioscaffold materials are essential for constructing tissue regeneration microenvironments. In this study, by using an electrospinning technique without any other additives, we successfully developed pure porcine decellularized nerve matrix (xDNME) conduits. The developed xDNME was composed of an obvious decellularized matrix fiber structure and effectively retained the natural components in the decellularized matrix of the nerve tissue. The xDNME conduit exhibited superior biocompatibility and the ability to overcome inter-species barriers. In vivo, after 12 weeks of implantation, xDNME significantly promoted the regeneration of rat sciatic nerve. The regenerated nerve fibers completely connected the two ends of the nerve defect, which were about 8 mm apart. The xDNME and xDNME-OPC groups showed myelin structures in the regenerated nerve fibers. In the xDNME group, the average thickness of the regenerated myelin sheath was 0.640 ± 0.013 µm, which was almost comparable to that in the autologous nerve group (0.646 ± 0.017 µm). Electrophysiological experiments revealed that both of the regenerated nerve fibers in the xDNME and xDNME-OPC groups had excellent abilities to transmit electrical signals. Respectively, the average conduction velocities of xDNME and xDNME-OPC were 8.86 ± 3.57 m/s and 6.99 ± 3.43 m/s. In conclusion, the xDNME conduits have a great potential for clinical treatment of peripheral nerve injuries, which may clinically transform peripheral nerve related regenerative medicine.

Co-culture of Schwann cells and endothelial cells for synergistically regulating dorsal root ganglion behavior on chitosan-based anisotropic topology for peripheral nerve regeneration.

Background: Anisotropic topologies are known to regulate cell-oriented growth and induce cell differentiation, which is conducive to accelerating nerve regeneration, while co-culture of endothelial cells (ECs) and Schwann cells (SCs) can significantly promote the axon growth of dorsal root ganglion (DRG). However, the synergistic regulation of EC and SC co-culture of DRG behavior on anisotropic topologies is still rarely reported. The study aims to investigate the effect of anisotropic topology co-cultured with Schwann cells and endothelial cells on dorsal root ganglion behavior for promoting peripheral nerve regeneration. Methods: Chitosan/artemisia sphaerocephala (CS/AS) scaffolds with anisotropic topology were first prepared using micro-molding technology, and then the surface was modified with dopamine to facilitate cell adhesion and growth. The physical and chemical properties of the scaffolds were characterized through morphology, wettability, surface roughness and component variation. SCs and ECs were co-cultured with DRG cells on anisotropic topology scaffolds to evaluate the axon growth behavior. Results: Dopamine-modified topological CS/AS scaffolds had good hydrophilicity and provided an appropriate environment for cell growth. Cellular immunofluorescence showed that in contrast to DRG growth alone, co-culture of SCs and ECs could not only promote the growth of DRG axons, but also offered a stronger guidance for orientation growth of neurons, which could effectively prevent axons from tangling and knotting, and thus may significantly inhibit neurofibroma formation. Moreover, the co-culture of SCs and ECs could promote the release of nerve growth factor and vascular endothelial growth factor, and up-regulate genes relevant to cell proliferation, myelination and skeletal development via the PI3K-Akt, MAPK and cytokine and receptor chemokine pathways. Conclusions: The co-culture of SCs and ECs significantly improved the growth behavior of DRG on anisotropic topological scaffolds, which may provide an important basis for the development of nerve grafts in peripheral nerve regeneration.

Development of a polyacrylamide/chitosan composite hydrogel conduit containing synergistic cues of elasticity and topographies for promoting peripheral nerve regeneration.

Substrate elasticity and topographical guidance are crucial factors for regulating tissue regeneration, but the synergistic effects of both cues on peripheral nerve regeneration are still unclear. In this paper, polyacrylamide/chitosan (PAM/CS) composite hydrogels with synergistic characteristics of elasticity and morphology were prepared using in situ free-radical polymerization and micro-molding. The physicochemical properties of hydrogels were characterized, and the effect on peripheral nerve regeneration was systematically evaluated via in vitro and in vivo experiments, respectively. The in vitro experiments showed that on a PAM/CS composite hydrogel with an elastic modulus of 5.822 kPa/8.41 kPa and a surface groove width of 30 µm, the dorsal root ganglion (DRG) neurite had a strong growth ability and better-oriented status. The samples were taken from each group at 2 and 12 weeks after bridging rabbit sciatic nerve defects with a PAM/CS composite hydrogel conduit. General observation of the rabbit body and transplanted nerve, nerve electro-physiological examination, muscle wet weight recovery rate detection and comparison, observation of sciatic nerve frozen section immunofluorescence staining and myelinated nerve fiber recovery rate comparison were used to evaluate the effect of nerve transplantation. The elastic modulus of 8.41 kPa and groove width of 30 µm were similar to those of the autograft group. At the same time, the signaling pathways, including the focal adhesion markers vinculin, p-FAK, and Rho A protein, referring to axon adhesion and extension, were initially revealed. In summary, our developed hydrogel implants containing synergistic cues of elasticity and topographies may provide a new and effective strategy for the treatment of peripheral nerve injury in the future.

Bionic microenvironment-inspired synergistic effect of anisotropic micro-nanocomposite topology and biology cues on peripheral nerve regeneration.

Anisotropic topographies and biological cues can simulate the regenerative microenvironment of nerve from physical and biological aspects, which show promising application in nerve regeneration. However, their synergetic influence on injured peripheral nerve is rarely reported. In the present study, we constructed a bionic microenvironment-inspired scaffold integrated with both anisotropic micro-nanocomposite topographies and IKVAV peptide. The results showed that both the topographies and peptide displayed good stability. The scaffolds could effectively induce the orientation growth of Schwann cells and up-regulate the genes and proteins relevant to myelination. Last, three signal pathways including the Wnt/ß-catenin pathway, the extracellular signal-regulated kinase/mitogen-activated protein pathway, and the transforming growth factor-ß pathway were put forward, revealing the main path of synergistic effects of anisotropic micro-nanocomposite topographies and biological cues on neuroregeneration. The present study may supply an important strategy for developing functional of artificial nerve implants.

The Influence of the Surface Topographical Cues of Biomaterials on Nerve Cells in Peripheral Nerve Regeneration: A Review.

The surface topographies of artificial implants including surface roughness, surface groove size and orientation, and surface pore size and distribution have a great influence on the adhesion, migration, proliferation, and differentiation of nerve cells in the nerve regeneration process. Optimizing the surface topographies of biomaterials can be a key strategy for achieving excellent cell performance in various applications such as nerve tissue engineering. In this review, we offer a comprehensive summary of the surface topographies of nerve implants and their effects on nerve cell behavior. This review also emphasizes the latest work progress of the layered structure of the natural extracellular matrix that can be imitated by the material surface topology. Finally, the future development of surface topographies on nerve regeneration was prospectively remarked.