Improved Efficacy of Delayed Treatment with Human Bone Marrow-Derived Stromal Cells Evaluated in Rats with Spinal Cord Injury.

The treatment of spinal cord injury (SCI) with uncultivated human bone marrow-derived stromal cells (bmSCs) prepared by negative selection has been proposed to be therapeutically superior to treatment with stem cells that were expanded in vitro. To explore their use in clinical trials, we studied the functional effects of delayed application at 7 days after SCI by testing different doses of bmSCs. Spinal cord contusion injury was induced in adult male Wistar rats at the thoracic level T9. Human bmSCs were prepared by negative selection without expansion in vitro (NeuroCellsTM). Treatment consisted of one 150 µL injection into the cisterna magna containing 0.5 or 2.5 million fresh bmSCs or 2.5 million bmSCs. The recovery of motor functions was evaluated during a surveillance period of six weeks (6 W), during which spinal cords were assessed histologically. Treatment resulted in a significant, dose-dependent therapeutic effect on the recovery of motor performance. The histological analysis revealed a lower degree of axonal degeneration and better survival of neurons and oligodendrocytes in bmSCs treated rats. Our results support delayed intrathecal application of bmSCs prepared by negative selection without expansion in vitro as a treatment of SCI.

Janus Kinase Inhibitor Brepocitinib Rescues Myelin Phagocytosis Under Inflammatory Conditions: In Vitro Evidence from Microglia and Macrophage Cell Lines.

Central nervous system (CNS) injuries induce cell death and consequently the release of myelin and other cellular debris. Microglia as well as hematogenous macrophages actively collaborate to phagocyte them and undergo their degradation. However, myelin accumulation persists in the lesion site long after the injury with detrimental effects on axonal regeneration. This might be due to the presence of inhibitors of phagocytosis in the injury site. As we recently published that some proinflammatory stimuli, like interferon-γ (IFNγ) and lipopolysaccharide (LPS), inhibit myelin phagocytosis in macrophages, we have now studied the signaling pathways involved. A phagocytosis assay in Raw264.7 macrophages and N13 microglia cell lines with labeled myelin was developed with the pHrodo reagent that emits fluorescence in acidic cellular compartments (e.g.lysosomes). Pharmacological inhibition of Janus kinases (Jak) with Brepocitinib restored myelin phagocytosis and rescued the expression of genes related to phagocytosis, like triggering receptor expressed on myeloid cells 2 (TREM2), induced by IFNγ or LPS. In addition, while pharmacological inhibition of the signal transducer and activator of transcription 3 (STAT3) rescued myelin phagocytosis and the expression of phagocytosis related genes in the presence of LPS, it did not have any effect on IFNγ-treated cells. Our results show that Jak pathways participate in the inhibition of myelin phagocytosis by IFNγ and LPS. They also indicate that the resolution of inflammation is important for the clearance of cellular debris by macrophages and subsequent regenerative processes.

Tauroursodeoxycholic Acid Reduces Neuroinflammation but Does Not Support Long Term Functional Recovery of Rats with Spinal Cord Injury.

The bile acid tauroursodeoxycholic acid (TUDCA) reduces cell death under oxidative stress and inflammation. Implants of bone marrow-derived stromal cells (bmSC) are currently under investigation in clinical trials of spinal cord injury (SCI). Since cell death of injected bmSC limits the efficacy of this treatment, the cytoprotective effect of TUDCA may enhance its benefit. We therefore studied the therapeutic effect of TUDCA and its use as a combinatorial treatment with human bmSC in a rat model of SCI. A spinal cord contusion injury was induced at thoracic level T9. Treatment consisted of i.p. injections of TUDCA alone or in combination with one injection of human bmSC into the cisterna magna. The recovery of motor functions was assessed during a surveillance period of six weeks. Biochemical and histological analysis of spinal cord tissue confirmed the anti-inflammatory activity of TUDCA. Treatment improved the recovery of autonomic bladder control and had a positive effect on motor functions in the subacute phase, however, benefits were only transient, such that no significant differences between vehicle and TUDCA-treated animals were observed 1-6 weeks after the lesion. Combinatorial treatment with TUDCA and bmSC failed to have an additional effect compared to treatment with bmSC only. Our data do not support the use of TUDCA as a treatment of SCI.

Bile acids attenuate PKM2 pathway activation in proinflammatory microglia.

Glycolysis is the metabolic pathway that converts glucose into pyruvate. Central nervous system (CNS) pathologies, such as spinal cord injury (SCI) and ischemia, are accompanied by an increase of the glycolytic pathway in the damaged areas as part of the inflammatory response. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenolpyruvate and ADP to pyruvate and ATP. The protein has two isoforms, PKM1 and PKM2, originated from the same gene. As a homodimer, PKM2 loses the pyruvate kinase activity and acts as a transcription factor that regulates the expression of target genes involved in glycolysis and inflammation. After SCI, resident microglia and hematogenous macrophages are key inducers of the inflammatory response with deleterious effects. Activation of the bile acid receptor TGR5 inhibits the pro-inflammatory NFκB pathway in microglia and macrophages. In the present study we have investigated whether bile acids affect the expression of glycolytic enzymes and their regulation by PKM2. Bacterial lipopolysaccharide (LPS) induced the expression of PKM1, PKM2 and its target genes in primary cultures of microglial and Raw264.7 macrophage cells. SCI caused an increase of PKM2 immunoreactivity in macrophages after SCI. Pretreatment with tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA) reduced the expression of PKM2 and its target genes in cell cultures. Similarly, after SCI, TUDCA treatment reduced the expression of PKM2 in the lesion center. These results confirm the importance of PKM2 in the inflammatory response in CNS pathologies and indicate a new mechanism of bile acids as regulators of PKM2 pathway.

Biofunctionalized PEDOT-coated microfibers for the treatment of spinal cord injury.

Poly(3, 4-ethylenedioxythiophene)-coated carbon microfibers (PEDOT-MFs) hold promise for developing advanced neuroprostheses and neural repair devices. We investigated the chronic cellular responses to PEDOT-MFs implanted into the uninjured and the transected rat spinal cord, and compared the effects of polymer surface biofunctionalization with covalently attached polylysine (PLL) or a multimolecular complex of PLL, heparin, basic fibroblast growth factor (bFGF), and fibronectin. An alginate gel was used to facilitate microfiber implantation and reduce connective tissue scarring after spinal cord injury (SCI). PLL/heparin/bFGF/fibronectin-functionalized PEDOT-MFs showed excellent integration within the uninjured and injured spinal cord, frequently establishing contact with neuronal somas, axons, dendrites and glial cells, accompanied by very little or absent scarring response. On the contrary, non-functionalized and PLL-functionalized microfibers provoked inflammation and fibrosis with loss of neural elements in the surrounding tissue. Within the lesion, the PEDOT-MFs by themselves facilitated longitudinal alignment of migratory cells and growing axons, and their modification with PLL/heparin/bFGF/fibronectin promoted tissue healing, enhancing blood vessel formation and axonal regeneration without increasing inflammation. These results support the incorporation of biofunctionalized electroconducting microfibers in neuro-electronic interfaces and lesion-bridging systems for the treatment of SCI.

Glial progenitor cell migration promotes CNS axon growth on functionalized electroconducting microfibers.

Electroactive systems that promote directional axonal growth and migration of glial progenitor cells (GPC) are needed for the treatment of neurological injuries. We report the functionalization of electroconducting microfibers with multiple biomolecules that synergistically stimulate the proliferation and migration of GPC, which in turn induce axonal elongation from embryonic cerebral cortex neurons. PEDOT doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] was synthesized on carbon microfibers and used for covalent attachment of molecules to the electroactive surface. The molecular complexes that promoted GPC proliferation and migration, followed by axonal extension, were composed of polylysine, heparin, basic fibroblast growth factor (bFGF), and matricellular proteins; the combination of bFGF with vitronectin or fibronectin being indispensable for sustained glial and axonal growth. The rate of glial-induced axonal elongation was about threefold that of axons growing directly on microfibers functionalized with polylysine alone. Electrical stimuli applied through the microfibers released bFGF and fibronectin from the polymer surface, consequently reducing GPC proliferation and promoting their differentiation into astrocytes, without causing cell detachment or toxicity. These results suggest that functionalized electroactive microfibers may provide a multifunctional tool for controlling neuron-glia interactions and enhancing neural repair. STATEMENT OF SIGNIFICANCE: We report a multiple surface functionalization strategy for electroconducting microfibers (MFs), in order to promote proliferation and guided migration of glial precursor cells (GPC) and consequently create a permissive substrate for elongation of central nervous system (CNS) axons. GPC divided and migrated extensively on the functionalized MFs, leading to fast elongation of embryonic cerebral cortex axons. The application of electric pulses thorough the MFs controlled glial cell division and differentiation. The functionalized MFs provide an advanced tool for neural tissue engineering and for controlling neuron-glial interactions. CNS axonal growth associated to migratory glial precursors, together with the possibility of directing glial differentiation by electrical stimuli applied through the MFs, open a new research avenue to explore for CNS repair.

Tailored Fringed Platforms Produced by Laser Interference for Aligned Neural Cell Growth.

Ordering neural cells is of interest for the development of neural interfaces. The aim of this work is to demonstrate an easy-to-use, versatile, and cost/time effective laser-based approach for producing platforms that promote oriented neural growth. We use laser interferometry to generate fringed channels with topography on partially reduced graphene oxide layers as a proof-of-concept substrate. We study cell adhesion, morphology, viability, and differentiation in cultures of embryonic neural progenitor cells on platforms with a 9.4 µm period. Results evidence that fringed platforms significantly promote neurite alignment (≈50% at 6 d), while preserving viability and neural differentiation.

Chondroitin sulphate-based 3D scaffolds containing MWCNTs for nervous tissue repair.

Nervous tissue lesions are an important social concern due to their increasing prevalence and their high sanitary costs. Their treatment still remains a challenge because of the reduced ability of nervous tissue to regenerate, its intrinsic structural and functional complexity and the rapid formation of fibroglial scars inhibiting neural repair. Herein, we show that 3D porous scaffolds made of chondroitin sulphate (CS), a major regulatory component of the nervous tissue, and multi-walled carbon nanotubes (MWCNTs) are selective substrates for the formation of a viable and neuron-enriched network with a transitory low glial content. Scaffolds have been fabricated by using the ice segregation-induced self-assembly technique and cultured with embryonic neural progenitor cells. Cell adhesion, morphology, viability, neuron/glial differentiation, calcium signaling dynamics, and mitochondrial activity have been studied over time on the scaffolds and compared to appropriate 2D control substrates. Our results indicate the formation of viable cultures enriched in neuron cells for up to 20 days, with ability to display calcium transients and active mitochondria, even in the absence of poly-D-lysine coating. A synergistic neural-permissive signaling from both the scaffold structure and its components (i.e., MWCNTs and CS) is suggested as the major responsible factor for these findings. We anticipate that these scaffolds may serve nerve regeneration if implanted in the acute phase after injury, as it is during the first stages of graft implantation when the most critical sequence of phenomena takes place to drive either nervous regeneration or fibroglial scar formation. The temporary glial inhibition found may be, indeed, beneficial for promoting the formation of neuron-enriched circuits at early phases while guaranteeing posterior glial integration to support longer-term neuron survival and activity.

N-Cadherin- and L1-functionalised conducting polymers for synergistic stimulation and guidance of neural cell growth.

Conducting polymers are promising materials for advanced neuroprostheses and neural repair devices. However, these challenging technologies demand stable presentation of multiple biomolecules on the polymer surface and fabrication of scaffolds suitable for implantation. We electrosynthesised poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) on gold-coated surfaces or carbon microfibres, functionalised the polymer by covalent immobilisation of anti-IgG antibodies and subsequent binding of N-Cadherin and L1 recombinant proteins, and used these materials as substrates for culturing cerebral cortex neurons. N-Cadherin and L1 were much more effective than polylysine in promoting axonal elongation and collateralisation on the polymer. However, N-Cadherin also induced cell migration and dendritic extension and branching, whereas L1 inhibited dendrites. Dual functionalisation with N-Cadherin and L1 produced synergistic effects on neuronal growth that could not be achieved with either of the proteins when used alone. PEDOT:PSS-co-MA electrosynthesised on carbon microfibres showed good electrochemical properties and, when biofunctionalised with N-Cadherin or L1, stimulated very long and guided axonal elongation. Finally, electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry showed that the good electrical properties of PEDOT:PSS-co-MA were not degraded by covalent peptide attachment, indicating that this polymer is suitable for multiple biofunctionalisation of electroactive surfaces in neuroprosthetic and lesion-bridging applications.

Bioelectrochemical control of neural cell development on conducting polymers.

Electrically conducting polymers hold promise for developing advanced neuroprostheses, bionic systems and neural repair devices. Among them, poly(3, 4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) exhibits superior physicochemical properties but biocompatibility issues have limited its use. We describe combinations of electrochemical and molecule self-assembling methods to consistently control neural cell development on PEDOT:PSS while maintaining very low interfacial impedance. Electro-adsorbed polylysine enabled long-term neuronal survival and growth on the nanostructured polymer. Neurite extension was strongly inhibited by an additional layer of PSS or heparin, which in turn could be either removed electrically or further coated with spermine to activate cell growth. Binding basic fibroblast growth factor (bFGF) to the heparin layer inhibited neurons but promoted proliferation and migration of precursor cells. This methodology may orchestrate neural cell behavior on electroactive polymers, thus improving cell/electrode communication in prosthetic devices and providing a platform for tissue repair strategies.

Nitric oxide elicits functional MMP-13 protein-tyrosine nitration during wound repair.

Nitric oxide (NO) plays a critical role in wound healing, in part by promoting angiogenesis. However, the precise repair pathways affected by NO are not well defined. We now show that NO regulates matrix metalloproteinase-13 (MMP-13) release during wound repair. We find that normally MMP-13 is kept inside endothelial cells by an association with caveolin-1. However, nitration of MMP-13 on tyrosine residue Y338 causes it to dissociate from caveolin-1 and be released from endothelial cells. We next explored the functional significance of MMP-13 nitration in vivo. Skin injury increases nitration of MMP-13 in mice. Skin wounds in inducible nitric oxide synthase knockout mice release less MMP-13 and heal more slowly than skin wounds in wild-type mice. Conversely, skin wounds in caveolin-1 knockout mice have increased NO production, increased MMP-13 nitration, and accelerated wound healing. Collectively, our data reveal a new pathway through which NO modulates wound repair: nitration of MMP-13 promotes its release from endothelial cells, where it accelerates angiogenesis and wound healing.

Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts.

During bone development, osteoblast differentiation requires remodeling of the extracellular matrix. Although underlying mechanisms have not been elucidated, evidence points to the participation of the nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) system. Here, we detected increased matrix metalloproteinase (MMP)-13 mRNA, protein and activity, as well as increased inducible NO synthase (iNOS) and NO production during the differentiation of MC3T3-E1 osteoblasts. Transcriptional activity of the MMP-13 promoter was augmented by NO, 8-bromo-cGMP (8-Br-cGMP), and by a dominant-positive form of protein kinase G (PKG1-alpha). The stimulatory effect on the MMP-13 promoter was partially inhibited by mutation of the osteoblast-specific element 2 (OSE-2) binding site. Core binding factor-1 (Cbfa-1) expression peaked at 7 days of differentiation, and was phosphorylated by PKG in vitro. Cbfa-1 was localized to cell nuclei, and its translocation was inhibited by the iNOS inhibitor 1400W. Immunohistological examination revealed that MMP-13 and Cbfa-1 expression levels are both reduced in 17-day-old embryos of iNOS-deficient mice. Silencing of Cbfa-1 mRNA blocked MMP-13 expression without interfering with endogenous NO production, confirming its role in NO-induced MMP-13 expression by MC3T3-E1 cells. The results described here suggest a mechanism by which NO regulates osteogenesis.