Hydrogels and cell based therapies in spinal cord injury regeneration

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (Grant no. PTDC/SAU-BMA/114059/2009; IF Development Grant to António J. Salgado); Prémios Santa Casa Neurociências for funds attributed to António J. Salgado under the scope of the Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)

In vitro characterization of injectable collagen and collagen-genipin hydrogels for neural tissue engineering

Nervous system injury leads to the permanent loss of sensory and motor functions. Injectable hydrogel containing therapeutic agents can be directly injected to the injured cavity as a promising approach for minimally-invasive treatment of nerve injury. However, such injectable hydrogels have not been well developed and documented in the literature. As inspired, this project aims to develop injectable collagen-based gels for nerve injury repair and to characterize in vitro for supporting neurite outgrowth of dorsal root ganglia (DRG) explants and dissociated neurons. To develop collagen-based gels, collagen at varying concentrations (e.g. 1.5, 2 and 2.5 mg/mL) were used to form gels under physiological conditions and genipin (0.25-5 mM) were applied as the chemical crosslinker. Characterization studies showed that collagen-based hydrogels could form porous and fibrillary gels within a time period of 40 s at 37 °C and genipin could significantly improve the mechanical property of gels and the resistance to degradation. To evaluate the cytotoxicity of the injectable hydrogels and compare the cell behaviour in two-dimensional (2D) and three-dimensional (3D) environments, rat primary Schwann cells (PRSCs) were seeded onto and encapsulated within the gels, and the cell viability was examined at Day 3 by the Live/Dead assay. The results showed that collagen gels provide superior support for PRSCs survival in both 2D and 3D cultures, for example, with a cell viability of 96 % and 95 %, respectively, for the collagen gel with a concentration of 1.5 mg/mL. Collagen chemically crosslinked by genipin at 0.25 and 0.5 mM exhibited a permissive but less favorable environment to PRSCs comparing with pure collagen. Genipin over 1 mM inhibited the PRSCs survival significantly in both 2D and 3D cultures. DRG explant and dissociated neuron cultures were examined as in vitro cell models to evaluate the therapeutic efficacy of collagen and collagen-genipin gels for nerve injury repair and the cellular response was also characterized and compared to each other. Preliminary 2D cultures were shown to greatly support neurite extension and 2.5 mg/mL collagen gel supported the most neurite extension and branching development. It was shown that genipin had a significant effect on the neurite density but not neurite length of DRG explants, whereas the dissociated neurons were more sensitive to genipin. Enrichment of culture medium with nerve growth factor (NGF) could significantly enhance the neurite length and density. PRSCs as the supportive cells were co-cultured with DRG explants/dissociated neurons in 3D hydrogels. Confocal microscopy showed that the neurites of DRG explants and dissociated neurons could extend freely within the physical collagen gels, and dissociated neurons exhibited pseudo-unipolar phenotype in 3D environment indicating true axonal extension. Moreover, genipin had a significantly inhibitory effect on dissociated neurons whereas the explants were more tolerant to genipin possibly due to the preserved cellular components and interactions. It was also shown that hydrogels infiltrated with PRSCs could enhance the neurite elongation and branches dramatically. Our research has determined the therapeutic potency of injectable collagen-based gels containing the PRSCs for nerve injury repair and gained new insights into the use of the injectable gel as a delivery substrate in neural tissue engineering

Sustained Dual Drug Delivery of Anti-Inhibitory Molecules for Spinal Cord Injury Treatment

Regeneration of lost synaptic connections following spinal cord injury (SCI) is limited due to local ischemia, cell death, and an excitotoxic environment, which leads to the development of an inhibitory glial scar surrounding a cystic cavity. Myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs) are major inhibitors to axon growth inhibition found within the glial scar and limit functional recovery. The NEP1-40 peptide competitively binds the Nogo receptor and partially blocks inhibition from MAIs, while chondroitinase ABC (ChABC) enzymatically digests CSPGs, which are upregulated at the site of injury. The first part of this work develops drug delivery systems which provide sustained delivery of both NEP1-40 and ChABC. In vitro studies showed that the combination of ChABC and NEP1-40 increased neurite extension compared to either treatment alone when dissociated embryonic dorsal root ganglia were seeded onto inhibitory substrates containing both MAIs and CSPGs. Furthermore, the ability to provide sustained delivery of biologically active ChABC and NEP1-40 from biomaterial scaffolds was achieved by loading ChABC into lipid microtubes and NEP1-40 into poly (lactic-co-glycolic acid) (PLGA) microspheres, obviating the need for invasive intrathecal pumps or catheters. Fibrin scaffolds embedded with the drug delivery systems (PLGA microspheres and lipid microtubes) were capable of releasing active ChABC for up to one week and active NEP1-40 for over two weeks in vitro. In addition, the loaded drug delivery systems in fibrin scaffolds decreased CSPG deposition and development of a glial scar, while also increasing axon growth after spinal cord injury in vivo. Therefore, the sustained, local delivery of ChABC and NEP1-40 within the injured spinal cord may block both myelin and CSPG-associated inhibition and allow for improved axon growth. The second part of this work looked to improve upon previously established therapies using a combination strategy. A variety of single therapy interventions provide small improvements in functional recovery after SCI but are limited due to the multitude of obstacles limiting recovery. Therefore, a multifactorial therapeutic option that combines several single therapies may provide a better chance of improving recovery. To this end, fibrin scaffolds were modified to provide sustained delivery of neurotrophic factors, the sustained delivery of anti-inhibitory molecules, and encapsulation of embryonic stem cell-derived progenitor motor neurons (pMNs). The efficacy of the scaffolds, prior to transplantation, was established by validating pMN viability, migration, and extension of processes was unaffected by culture within scaffolds with sustained delivery of anti-inhibitory molecules. The combination scaffolds were then transplanted into a rat sub-acute SCI model. The anti-inhibitory molecules were capable of removing proteoglycans within the glial scar when embedded in fibrin scaffolds without pMNs included. While pMNs incorporated into fibrin scaffolds without anti-inhibitory molecules showed significant cell survival, differentiation into neuronal cell types, axonal extension in the transplant area, and the ability to integrate into the host tissue, the combination of pMNs with anti-inhibitory molecules led to decreased cell survival and increased inflammation in the lesion site. Thus combination therapies maintain therapeutic potential for treatment of SCI but further work is needed to improve cell survival and limit inflammation

Forever young: How to control the elongation, differentiation, and proliferation of cells using nanotechnology

Within the emerging field of stem cells there is a need for an environment that can regulate cell activity, to slow down differentiation or proliferation, in vitro or in vivo while remaining invisible to the immune system. By creating a nanoenvironment surrounding PC12 cells, Schwann cells, and neural precursor cells (NPCs), we were able to control the proliferation, elongation, differentiation, and maturation in vitro. We extended the method, using self-assembling nanofiber scaffold (SAPNS), to living animals with implants in the brain and spinal cord. Here we show that when cells are placed in a defined system we can delay their proliferation, differentiation, and maturation depending on the density of the cell population, density of the matrix, and the local environment. A combination of SAPNS and young cells can be implanted into the central nervous system (CNS), eliminating the need for immunosuppressants. Copyright © 2009 Cognizant Comm. Corp.published_or_final_versio

Combinational treatment approach for traumatic spinal cord injury

Indiana University-Purdue University Indianapolis (IUPUI)Spinal cord injury (SCI) is devastating and debilitating, and currently no effective treatments exist. Approximately, 12,000 new cases of SCI occur annually in the United States alone. The central nervous system has very low repair capability after injury, due to the toxic environment in the injured tissue. After spinal cord trauma, ruptured blood vessels cause neighboring cells and tissues to be deprived of oxygen and nutrients, and result in the accumulation of carbon dioxide and waste. New blood vessels form spontaneously after SCI, but then retract as the injured tissue forms a cavity. Thus, the newly formed vasculature likely retracts because it lacks a structural support matrix to extend across the lesion. Currently, in the field of spinal cord injury, combinational treatment approaches appear to hold the greatest therapeutic potential. Therefore, the aim of these studies was to transplant a novel, non-immunogenic, bioengineered hydrogel, into the injured spinal cord to serve as both a structural scaffold (for blood vessels, axons, and astrocytic processes), as well as a functional matrix with a time-controlled release of growth factors (Vascular endothelial growth factor, VEGF; Glial cell line-derived neurotrophic factor, GDNF). The benefit of this hydrogel is that it remains liquid at cooler temperatures, gels to conform to the space surrounding it at body temperature, and was designed to have a similar tensile strength as spinal cord tissue. This is advantageous due to the non-uniformity of lesion cavities following contusive spinal cord injury. Hydrogel alone and combinational treatment groups significantly improved several measures of functional recovery and showed modest histological improvements, yet did not provoke any increased sensitivity to a thermal stimulus. Collectively, these findings suggest that with further investigation, hydrogel along with a combination of growth factors might be a useful therapeutic approach for repairing the injured spinal cord

Erratum to: Mechano-Transduction Signals Derived from Self-Assembling Peptide Nanofibers Containing Long Motif of Laminin Influence Neurogenesis in In-Vitro and In-Vivo (Mol Neurobiol, 10.1007/s12035-016-9836-z)

Astroglial scaring and limited neurogenesis are two problematic issues in recovery of spinal cord injury (SCI). In the meantime, it seems that mechanical manipulations of scaffold to inhibit astroglial scarring and improve neurogenesis is worthy of value. In the present investigation, the effect of nanofiber (gel) concentration as a mechanical-stimuli in neurogenesis was investigated. Cell viability, membrane damage, and neural differentiation derived from endometrial stem cells encapsulated into self-assembling peptide nanofiber containing long motif of laminin were assessed. Then, two of their concentrations that had no significant difference of neural differentiation potential were selected for motor neuron investigation in SCI model of rat. MTT assay data showed that nanofibers at the concentrations of 0.125 and 0.25 % w/v induced higher and less cell viability than others, respectively, while cell viability derived from higher concentrations of 0.25 % w/v had ascending trend. Gene expression results showed that noggin along with laminin motif over-expressed TH gene and the absence of noggin or laminin motif did not in all concentrations. Bcl2 over-expression is concomitant with the decrease of nanofiber stiffness, NF+ cells increment, and astrogenesis inhibition and dark neuron decrement in SCI model. It seems that stiffness affects on Bcl2 gene expression and may through β-Catenin/Wnt signaling pathway and BMP-4 inhibition decreases astrogenesis and improves neurogenesis. However, stiffness had a significant effect on upregulation of GFAP+ cells and motor neuron recovery in in vivo. It might be concluded that eventually there is a critical definitive point concentration that at less or higher than of it changes cell behavior and neural differentiation through different molecular pathways

COVALENT IMMOBILIZATION OF L1 NEURAL CELL ADHESION MOLECULE TO ACRYLATED TETRONIC® HYDROGELS FOR NEURAL REGENERATION

Spinal cord injuries cost the United States $20 billion per year, with an existing patient population of 256,000 growing by an estimated 12,000 each year. Current clinical therapies for spinal cord injury are limited to spinal immobilization and realignment via traction, surgery, administration of methylprednisolone sodium succinate (MPSS) within eight hours post-injury, and rehabilitation exercises. While these therapies are important and may minimize damage and restore limited function, there is a dire clinical need for treatments to address the growing population of chronically-injured patients. Varying degrees of axonal regeneration and functional recovery following spinal cord injury have been achieved in animal models by transplantation of glial cells from the peripheral nervous system and olfactory region. The recent identification of bioactive soluble and adhesive molecules produced by glial cells provides the opportunity to deliver these stimuli through biomaterial-mediated approaches, such as controlled release, gene therapy, and recombinant protein immobilization. The long-term objective of this project is a biomimetic, multi-factorial approach utilizing grooved fibers to restore structure and provide guidance for regenerating axons coupled with bioactive adhesive molecule delivery via immobilization to a hydrogel within the fiber grooves and controlled release of neurotrophic factors from the hydrogel. The implant design can serve as a platform for both in vitro and in vivo analysis of combination therapies for different injured nerve populations. The first part of this research focused on cloning and expression of a bioactive 140kDa fragment of L1 neural cell adhesion molecule. L1 is a particularly attractive candidate for neural regeneration because it is critical for proper nervous system development and in vitro studies have demonstrated selectivity of neuron adhesion to L1 in the presence of astrocytes, which play a major role in nervous system inflammation. The second part of this research focused on the synthesis and purification of acrylated Tetronic macromers, and the development of Michael addition methods for hydrogel crosslinking and protein immobilization. In order to establish the feasibility of these hydrogels for neural regeneration, initial testing was conducted using NIH 3T3 fibroblasts and fibronectin because of the well-known RGD-dependent interaction. Results demonstrated that fibronectin encapsulation and surface-immobilization through acrylation of fibronectin positively influenced fibroblast spreading and proliferation. The last part of this research focused on evaluating neuronal cell line and primary neuron response to L1, evaluating cytocompatibility of T904-acrylate hydrogels with neural cells, and developing immobilization methods for L1. Results indicate that surface-immobilization of L1 to hydrogels may be the most promising method of bioactive cell adhesion molecule delivery for neural regeneration

CAPILLARY CHANNEL POLYMER FIBER-BASED SCAFFOLDS FOR NEURAL REGENERATION

Over 50 million Americans are affected by ailments to the central nervous system (CNS) and it impacts the American economy over $400 billion a year. The number of people in the United States who have spinal cord injury (SCI) has been estimated to be approximately 276,000 persons as of 2014 with a range from 240,000 to 337,000 persons. The annual incidence of SCI, not including those who die at the scene of the accident is approximately 12,500 new cases each year. Due to the limited regenerative capacity of the adult CNS and lack of clinically effective therapies, these conditions commonly result in permanent functional deficits. SCI damages both ascending sensory and descending motor axonal pathways interrupting the transmission of synaptic signals between the brain and peripheral tissues. Although damaged axons attempt an initial regenerative response, this is rapidly aborted due to the presence of growth inhibitory molecules in CNS myelin and the glial scar and intrinsic limitations of adult CNS neuronal biochemistry such as the ability to maintain cAMP levels and upregulate the expression of `regeneration-associated genes\u27. On the other hand, TBI, stroke, and Parkinson\u27s disease result in neuronal cell death. The CNS has limited capacity to replace lost neurons because the neurons themselves are terminally differentiated and post-mitotic. Although neural stem cells (NSCs) have been identified in specialized regions of the adult brain such as the sub-ventricular zone (SVZ) and the sub-granular zones (SGZ), their number is insufficient and the pathological environment inadequate to support an effective regenerative response. The end goal of this project is to develop a biomimetic scaffold using grooved fibers for neural regeneration. This goal was met with a two-pronged approach. In the first approach, grooved fibers immobilized with bioactive adhesive molecule were developed to topographically guide regenerating axons. In the second approach, grooved fiber staples were used as cell-laden microcarriers and integrated into a composite hydrogel which demonstrated its ability to serve as a platform for cell proliferation. This latter approach can be translated into an injectable in situ crosslinkable scaffold that can be used for neural stem cell (NSC) delivery with the prospect of stem cell differentiation into neurons to replenish cell loss. The first part of this research focused on immobilizing a bioactive 140 kDa fragment of L1 neural cell adhesion molecule on uniquely designed groovy capillary channel polymer (CCP) fibers. L1-CAM is an attractive candidate for growth of spared axonal growth cones upon injury. It mediates CNS maturation, by means of neurite outgrowth, adhesion, fasciculation, migration, survival, myelination, axon guidance, synaptic plasticity and regeneration after trauma. High levels of L1 are expressed by growing axons during development and after SCI and there is a positive correlation between their expression and axonal growth. CCP fibers with surface immobilized L1-CAM were demonstrated to guide growth of primary neurons in vitro. In the latter part of this research, a methodology to fabricate CCP fiber staples was developed and these were employed as cell-laden microcarriers. These microcarriers were then integrated into a composite hydrogel blend and demonstrated high cell proliferation in vitro compared to control gels. This composite system can be a promising platform for NSC delivery and differentiation into neurons

Biomaterial implants combined with cell therapy improve axonal regeneration after spinal cord injury

Injury to the adult spinal cord damages ascending and descending spinal fiber tracts thereby disrupting proper information transmission between the brain, spinal cord and periphery of the body. Restoring neural connectivity beyond the site of injury is the essential prerequisite for functional recovery to occur. Without intervention, central nervous system (CNS) axons fail to regenerate, resulting in tremendous impairment of sensorimotor function as well as autonomic dysfunction and, consequently, a significant reduction of the patients’ quality of life. Hence, a massive effort has been spent to develop effective repair strategies for spinal cord injury (SCI) including cell transplantation and biomaterial implantation. However, functional axonal growth past the lesion site remains insufficient due to inappropriate implant integration, detri-mental fibroglial scarring and failure of spinal axons to grow beyond the site of injury. Recently, astrocytes were identified as essential key players for neuroregeneration due to their neuro-protective and supportive functions after CNS injury. Further, immature astrocytes not only fulfil scaffolding functions during development, but might also adapt to the harsh lesion envi-ronment without adopting detrimental phenotypes. Thus, astrocyte are prime candidates to provide structural as well as trophic support for growing axons in combination with biomaterial implants at SCI lesion sites. In the present study, novel alginate-based hydrogel implants with a defined channel micro-structure were combined with cellular grafts of immature astrocytes derived either from the cortex or the spinal cord of neonatal Fischer-344 rats to: (1) provide a physical guidance structure for regrowing axons at the site of injury; and (2) establish a permissive cellular growth substrate within and beyond the hydrogel implant supporting axonal crossing of the lesion cavity of a cervical unilateral hemisection of the spinal cord in adult rats. First, alginate-based hydrogel implants were modified with polypetides to improve their bio-compatibility and cell viability in vitro and in vivo. Afterwards, immature astrocytes from neona-tal rat cortex were cultivated and enriched in vitro. Seeding of alginate-based hydrogel im-plants with immature cortex-derived astrocytes improved axonal regrowth compared to non-seeded hydrogel implants following SCI. The grafted astrocytes interacted with the host as-trocytic network and aligned into tissue bridges structurally guiding axons across the host-graft interface. To elucidate whether astrocytes with a spinal cord identity would elicit superior pro-regenerative effects after SCI, immature astrocytes were isolated from the spinal cord of neonatal rats and compared with cortex-derived astrocytes. Phenotypic characterization re-vealed minor molecular and morphological differences between both astrocyte populations in vitro and in vivo. Particularly, cortex-derived astrocytes were found to have a more mature phenotype compared to spinal cord-derived astrocytes in vitro, however, both cell populations adopted a differentiated morphology and expressed functional molecular astrocytic markers in vivo after transplantation into the intact spinal cord. After SCI, seeded hydrogel implants to-gether with additional caudal grafts of either immature astrocyte population further enhanced axonal growth through the implantation site and promoted revascularization. The grafted cells connected with the host spinal parenchyma facilitating tissue bridging between implant and host. Finally, seeded hydrogel implants in combination with rostral and caudal immature astro-cyte grafts were shown to additionally increase axonal growth through the hydrogel implants after SCI by 70% compared to the previous transplantation paradigms. Thus, the combination of biomaterial implantation with cell transplantation superiorly promotes axonal growth through sites of acute SCI compared to treatment paradigms based only upon biomaterial implants. Moreover, additional grafts of immature astrocytes into the surrounding host tissue improve host-graft interactions by formation of a continuous cellular substrate spanning the SCI lesion site. Nonetheless, axonal re-entry into the distal host spinal cord may require additional trophic attraction