Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries.

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Recent therapeutic approaches for spinal cord injury.

A spinal cord injury (SCI) often causes permanent changes in strength and sensation functions below the site of the injury and affects thousands of people each year. Transplantation of stem cells is a promising approach in acute SCI as it may support spinal cord repair. However, in case of chronic SCI greater amounts of nervous tissue have to be regenerated, leaving scaffold transplantation the only feasible option for cellular engraftment and nervous bridging. The aim of regenerative medicine, specifically tissue engineering, is to create a microenvironment that mimics native extracellular matrix (ECM), capable of promoting specific cell-matrix interactions, coaxing cell behavior, and fostering host tissue regeneration. In this regard, nanostructured scaffolds are currently the most promising advanced substrates capable of supporting nervous fiber ingrowth and delivery of neurotrophic drugs. Among them, electrospinning technique and Self-Assembling Peptides (SAPs) have recently attracted lots of attention for their reproducible synthesis and high tailorability. This review highlights clinical trials and recent encouraging strategies for spinal cord repair comprising both cell therapy and nanomedicine.

Biomimetic electrospun PVDF/self-assembling peptide piezoelectric scaffolds for neural stem cell transplantation in neural tissue engineering.

Piezoelectric materials can provide in situ electrical stimulation without external chemical or physical support, opening new frontiers for future bioelectric therapies. Polyvinylidene fluoride (PVDF) possesses piezoelectricity and biocompatibility, making it an electroactive biomaterial capable of enhancing bioactivity through instantaneous electrical stimulation, which indicates significant potential in tissue engineering. In this study, we developed electroactive and biomimetic scaffolds made of electrospun PVDF and self-assembling peptides (SAPs) to enhance stem cell transplantation for spinal cord injury regeneration. We investigated the morphology and crystalline polymorphs of the electrospun scaffolds. Morphological studies demonstrated the benefit of using mixed sodium dodecyl sulfate (SDS) and SAPs as additives to form thinner, uniform, and defect-free fibers. Regarding electroactive phases, ß and γ phases-evidence of electroactivity-were predominant in aligned scaffolds and scaffolds modified with SDS and SAPs. In vitro studies showed that neural stem cells (NSCs) seeded on electrospun PVDF with additives exhibited desirable proliferation and differentiation compared to the gold standard. Furthermore, the orientation of the fibers influenced scaffold topography, resulting in a higher degree of cell orientation in fiber-aligned scaffolds compared to randomly oriented ones.

Biomimetic Electrospun Self-Assembling Peptide Scaffolds for Neural Stem Cell Transplantation in Neural Tissue Engineering.

Spinal cord regeneration using stem cell transplantation is a promising strategy for regenerative therapy. Stem cells transplanted onto scaffolds that can mimic natural extracellular matrix (ECM) have the potential to significantly improve outcomes. In this study, we strived to develop a cell carrier by culturing neural stem cells (NSCs) onto electrospun 2D and 3D constructs made up of specific crosslinked functionalized self-assembling peptides (SAPs) featuring enhanced biomimetic and biomechanical properties. Morphology, architecture, and secondary structures of electrospun scaffolds in the solid-state and electrospinning solution were studied step by step. Morphological studies showed the benefit of mixed peptides and surfactants as additives to form thinner, uniform, and defect-free fibers. It has been observed that ß-sheet conformation as evidence of self-assembling has been predominant throughout the process except for the electrospinning solution. In vitro NSCs seeded on electrospun SAP scaffolds in 2D and 3D conditions displayed desirable proliferation, viability, and differentiation in comparison to the gold standard. In vivo biocompatibility assay confirmed the permissibility of implanted fibrous channels by foreign body reaction. The results of this study demonstrated that fibrous 2D/3D electrospun SAP scaffolds, when shaped as micro-channels, can be suitable to support NSC transplantation for regeneration following spinal cord injury.

Mimicking Extracellular Matrix via Engineered Nanostructured Biomaterials for Neural Repair.

Extracellular matrix (ECM) consists of proteins, proteoglycans, and different soluble molecules. ECM provides structural support to mammalian cells. ECM is responsible for important cell functions, as well as assembling cells into various tissues and organs, regulating growth and cell-cell interaction. Recent studies have shown the potential of nanostructured biomaterials to mimic native ECM. Developing tailor-made biomaterials that mimic the complex nanoscale mesh of local ECM is not a trivial endeavor: bio-inspired biomaterials are designed to supply a healthy ECMlike structure, capable of filling the lesion cavity, favoring transplanted cell engraftment, providing physical support to endogenous neurogenesis and also tuning the inflammatory response to protect spared neurons. The strategies used to manufacture biomimetic hydrogel scaffold represent particularly important prospects of novel therapies for CNS regeneration. During this review, we describe with details the most promising regulatory pathways from ECM involved in the CNS injury and regeneration and we draw a line to the biomimetic potential of engineered nanostructured biomaterials aimed at mimicking extracellular matrix constructs and favoring the release of pro-regenerative agents. Lastly, a brief overview of their application in clinical trials is provided.

Self-assembling peptide hydrogels for the stabilization and sustained release of active Chondroitinase ABC in vitro and in spinal cord injuries.

Activated microglia/macrophages infiltration, astrocyte migration, and increased production of inhibitory chondroitin sulfate proteoglycans (CSPGs) are standard harmful events taking place after the spinal cord injuries (SCI). The gliotic scar, viz. the outcome of chronic SCI, constitutes a long-lasting physical and chemical barrier to axonal regrowth. In the past two decades, various research groups targeted the hostile host microenvironments of the gliotic scar at the injury site. To this purpose, biomaterial scaffolds demonstrate to provide a promising potential for nervous cell restoration. We here focused our efforts on two self-assembling peptides (SAPs), featuring different self-assembled nanostructures, and on different methods of drug loading to exploit the neuroregenerative potential of Chondroitinase ABC (ChABC), a thermolabile pro-plastic agent attenuating the inhibitory action of CSPGs. Enzymatic activity of ChABC (usually lasting less than 72 hours in vitro) released from SAPs was remarkably detected up to 42 days in vitro. ChABC was continuously released in vitro from a few days to 42 days as well. Also, injections of ChABC loaded SAP hydrogels favored host neural regeneration and behavioral recovery in chronic SCI in rats. Hence, SAP hydrogels showed great promise for the delivery of Chondroitinase ABC in future therapies targeting chronic SCI.

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury.

AIMS: Usually, spinal cord injury (SCI) develops into a glial scar containing extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABC (ChABC), from Proteus vulgaris degrading the glycosaminoglycan (GAG) side chains of CSPGs, offers the opportunity to improve the final outcome of SCI. However, ChABC usage is limited by its thermal instability, requiring protein structure modifications, consecutive injections at the lesion site, or implantation of infusion pumps. METHODS: Aiming at more feasible strategy to preserve ChABC catalytic activity, we assessed various stabilizing agents in different solutions and demonstrated, via a spectrophotometric protocol, that the 2.5 mol/L Sucrose solution best stabilized ChABC as far as 14 days in vitro. RESULTS: ChABC activity was improved in both stabilizing and diluted solutions at +37°C, that is, mimicking their usage in vivo. We also verified the safety of the proposed aqueous sucrose solution in terms of viability/cytotoxicity of mouse neural stem cells (NSCs) in both proliferating and differentiating conditions in vitro. Furthermore, we showed that a single intraspinal treatment with ChABC and sucrose reduced reactive gliosis at the injury site in chronic contusive SCI in rats and slightly enhanced their locomotor recovery. CONCLUSION: Usage of aqueous sucrose solutions may be a feasible strategy, in combination with rehabilitation, to ameliorate ChABC-based treatments to promote the regeneration of central nervous system injuries.

Recovery from experimental parkinsonism by intrastriatal application of erythropoietin or EPO-releasing neural precursors.

An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP-induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 105 erythropoietin-releasing neural precursor cells (Er-NPCs) rescued degenerating striatal dopaminergic neurons and promoted behavioral recovery as shown by three independent behavioral tests. These effects were replicated through direct intrastriatal administration of recombinant human EPO. At the end of the observational period, most of the transplanted Er-NPCs were vital and migrated via the striatum to reach Substantia Nigra. The restorative effects appear to be mediated by EPO since co-injection of anti-EPO or anti-EPOR antibodies antagonized the positive outcomes. Furthermore, this report supports the neuroprotective action of EPO, which may also be achieved via administration of EPO-releasing cells such as Er-NPCs.