Alexandre Leite Rodrigues de Oliveira

Highly porous hydrogel scaffolds are critical for advancing three-dimensional printing in tissue engineering. Interconnected porous networks facilitate nutrient diffusion, waste removal, and cell infiltration, necessary processes for tissue regeneration. Emulgel-based inks are developed using oil-in-water Pickering emulsions, in which mineral oil serves as a sacrificial porogen within a carboxymethyl cellulose (CMC)/Laponite matrix, with Laponite providing stabilization of the emulsion. Nine formulations are prepared by varying CMC (0.5–1.0 wt %) and mineral oil (10–20 wt %) concentrations at fixed Laponite (2 wt %). Rheological characterization confirms pronounced shear thinning, appropriate viscoelasticity, and rapid thixotropic recovery, establishing suitability for extrusion-based printing. Post-printing lyophilization and oil removal produce interconnected, homogeneous pore networks, as revealed by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Hydrated scaffolds exhibit compressive moduli of 20–30 kPa, overlapping with native soft tissue stiffness. Swelling and degradation analyses confirm structural stability under physiological conditions. Biological assessment using human dental pulp mesenchymal stromal cells (DPMSCs) identifies the 1.0 wt % CMC, Laponite, and 10 wt % oil formulation as optimal, supporting sustained cell proliferation over 28 days following lyophilization and pre-conditioning. This platform provides fabrication advances in 3D-printed hydrogel scaffolds, enhanced porosity, and mechanical properties for soft-tissue engineering and drug delivery applications.

Peripheral nerve transection requires surgical treatment, although functional recovery remains incomplete. Here, we developed a biofunctional nanocomposite nerve interface combining an ion‐covalent gelatin methacryloyl (GelMA)/Laponite hydrogel with engineered thermostable fibroblast growth factor‐2 (FGF‐2) to recreate a regenerative microenvironment. Laponite incorporation (2%–2.5% w/v) precisely modulated the hydrogel network, providing shear‐thinning behavior, enhanced viscoelasticity, microporosity, a twofold increase in compressive modulus, controlled degradation, injectability, and three‐dimensional (3D) bioprintability. Functionalization with thermostable FGF‐2 enabled sustained release while preserving bioactivity, promoting Schwann cell viability, metabolic activity, cytoskeletal organization, and upregulation of nerve growth factor receptor (NGFR), glial cell line‐derived neurotrophic factor (GDNF), early growth response protein 2 (EGR2), octamer‐binding transcription factor 6 (OCT6), and SRY‐box transcription factor 9 (SOX9). Nerve interfaces consisting of a basal polycaprolactone (PCL) layer and a biofunctional GelMA (10%)/Laponite (2%) hydrogel containing FGF‐2 (1 µg mL ^{− 1} ) were fabricated by 3D bioprinting followed by visible‐light crosslinking. In a rat neurotmesis model, FGF‐2‐functionalized wraps significantly improved motor function, electrophysiological recovery, Schwann cell activity (S100 calcium‐binding protein), neurofilament organization and myelination (FluoroMyelin), and muscle preservation. Collectively, this work introduces a 3D‐bioprinted, cell‐instructive nerve interface integrating mechanical neuroprotection with sustained neurotrophic signaling to enhance peripheral nerve regeneration.

Epidural fibrosis and insufficient bone regeneration following laminectomy remain an unresolved clinical problem. Artificial laminate has been proposed to address post-surgical complications by establishing a barrier between soft and mineralized tissues while providing spinal mechanical support. Herein, we developed and evaluated three-dimensional (3D) bioprinted polycaprolactone (PCL)/gelatin methacryloyl (GelMA) biocomplexes functionalized with either human dental pulp stem cells (hDPSCs) or thermostable fibroblast growth factor-2 (FGF-2) to address post-laminectomy tissue regeneration and fibrosis. In vitro characterization identified an optimal GelMA formulation (10%) exhibiting pseudoplastic behavior, sustained cell viability, and enhanced mineralized matrix deposition. Four in vivo experimental groups (n = 5/group) were analyzed: laminectomy without treatment (LWT), biocomplex alone (BC), BC+FGF-2, and BC+hDPSC. All implanted biocomplexes were well-tolerated, with preserved motor function and no evidence of radiological changes. Histological and immunohistochemical analyses revealed that all biocomplex groups significantly reduced the expression of fibrotic markers (MMP-2, MMP-9, vimentin) when compared to the LWT group (p < 0.05). Functionalized groups showed superior outcomes: BC+FGF-2 enhanced tissue organization and vascularization, while BC+hDPSC produced lamellar bone trabeculae with hematopoietic tissue. These results demonstrate that bioactive functionalization establishes pro-regenerative microenvironments that actively modulate post-surgical healing beyond a passive barrier function. Ultimately, these bioactive biomaterials hold strong translational potential for the functional restoration of spinal surgical defects.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease characterized by demyelination, neurodegeneration, and progressive neurological disability. Galanin, a neuropeptide with immunomodulatory properties, signals through G protein-coupled receptors, among which galanin receptor 2 (GALR2) has been implicated with neuroprotective and anti-inflammatory functions. A rare homozygous single nucleotide variant in GALR2 (rs61745847; p.W249L) has been identified in a patient diagnosed with relapsing-remitting MS, however, the biological relevance of this variant in neuroinflammation remains unknown. Here, we investigated the impact of the orthologous GALR2 W248L mutation using a knock-in mouse model and experimental autoimmune encephalomyelitis (EAE). GALR2 W248L knock-in (KI) mice exhibited a more severe clinical course of EAE, accompanied by enhanced inflammatory infiltration, exacerbated demyelination, and increased microglial activation in the spinal cord compared with wild-type (WT) mice. Despite comparable lymphoid and myeloid cell frequencies in the central nervous system, alterations in microglial density and morphology suggested an important contribution of the innate immune system to disease exacerbation in the KI mice. Ex vivo analyses revealed that bone marrow-derived macrophages from KI animals exhibited a pronounced shift toward a pro-inflammatory phenotype, characterized by enhanced M1 polarization, impaired M2-associated responses, and increased NLRP3 inflammasome activation. In parallel, live-cell imaging of primary hippocampal neurons demonstrated reduced galanin binding in mutant cells, consistent with impaired GALR2 functional availability at the plasma membrane. Together, these findings identify GALR2 as a modulator of the neuroinflammatory response and indicate that disruption of galanin-GALR2 signaling promotes sustained innate immune activation, highlighting the relevance of this pathway for MS pathogenesis and its potential as a therapeutic target in neuroinflammatory disorders.

Leishmania amazonensis is an intracellular protozoan parasite and etiological agent of Localised Cutaneous Leishmaniasis (LCL). A challenge in managing LCL is the frequent occurrence of secondary bacterial co‐infections caused by opportunistic bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus . This study aimed to evaluate the effects of Bacterial Membrane Vesicles (BMVs) from these bacteria on macrophages infected with L. amazonensis . BMVs were characterised by Nanoparticle Tracking Analysis (NTA), and their effects on macrophage viability, infection dynamics, and cytokine production were assessed. Macrophages infected with L. amazonensis and subsequently treated with BMVs exhibited reduced parasite burden and cell viability compared to infected controls. The production of cytokines was variably modulated depending on the vesicle source. TNF‐α levels were higher in macrophages infected with L. amazonensis and treated with BMVs than in those infected with the parasite alone. Additionally, elevated levels of IL‐1β were observed in co‐cultures treated with P. aeruginosa membrane vesicles (POMVs), suggesting a stronger inflammatory profile than that induced by S. aureus extracellular vesicles (SEVs). In contrast, IL‐4 and IL‐6 levels were not significantly affected by either POMVs or SEVs. These results indicate that uninfected and L. amazonensis ‐infected macrophages respond distinctly to POMVs and SEVs, particularly through enhanced secretion of IL‐1β and TNF‐α. In conclusion, macrophages exposed to both L. amazonensis and bacterial vesicles simulate a host–parasite–bacteria interaction, activating production of cytokines. Given their immunogenic properties, BMVs may represent promising targets for the development of therapeutic strategies associated with LCL.

Spinal root injuries trigger longitudinal spinal cord damage, leading to motoneuron degeneration, gliosis, and synaptic loss. Glutamate excitotoxicity through NMDA and AMPA receptor overstimulation is a key driver of this pathology, highlighting NMDA receptor antagonists as potential neuroprotective agents. Here, we evaluated the effects of memantine after unilateral L4–L6 ventral root crush (VRC) in adult C57BL/6JUnib mice. Animals received daily oral gavage of vehicle or memantine (30, 45, or 60 mg/kg) for 14 days. At 28 days post‐injury, histological analysis showed that memantine reduced astrogliosis and microglial activation, while enhancing motoneuron survival (most pronounced at 45 mg/kg, p < 0.001) and preserving synaptic coverage ( p < 0.01), without significant changes in VGLUT‐1 or GAD65 expression. Consistently, RT‐qPCR analysis revealed early upregulation of inflammatory markers ( Ccr2, Itgam ) in vehicle‐treated mice, which was attenuated by memantine at 3–7 days post‐injury ( p < 0.05). These findings indicate that memantine confers neuroprotection in VRC by modulating inflammatory gene expression, mitigating gliosis, and promoting motoneuron survival, supporting its therapeutic potential in spinal cord injuries. image

Background: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by the absence of functional dystrophin, leading to progressive muscle degeneration, inflammation, and alterations in the central nervous system. The sustained inflammatory response in DMD increases glial activation and the release of tumor necrosis factor alpha (TNF-α), which contributes to muscle fiber damage. Here, we investigated the anti-inflammatory and neuroprotective effects of Etanercept, a TNF-α receptor-blocking therapeutic, on the spinal cord of MDX mice, a widely used model of DMD. Methods: Adult male MDX and control C57BL/10 mice received vehicle or Etanercept (3, 6, or 12 mg/Kg, intraperitoneally (i.p.)) every 72 h for two weeks, along with daily gait assessment. At the end of treatment, flow cytometry and immunolabeling analyses were performed in the lumbar spinal cord. Results: Etanercept at 12 mg/Kg reduced astrogliosis and microglial activation; restored synaptic markers, including synaptophysin, glutamic acid decarboxylase 65 (GAD-65), and vesicular glutamate transporter 1 (VGLUT-1); and decreased pro-inflammatory cytokines. The treatment reduced GFAP+/TNF-α+ astrocytes and significantly downregulated Th1 lymphocyte polarization in treated MDX mice. These cellular effects were accompanied by improvements in locomotor function. Conclusions: Together, our findings indicate that TNF-α blockade by Etanercept exerts neuroprotective and anti-inflammatory actions in the spinal cord of dystrophic mice, providing new insights into the impact of TNF-α signaling on neuroinflammatory processes in DMD.

Neurotmesis, the most severe form of peripheral nerve injury, involves complete transection and loss of motor and sensory function. Surgical repair, typically via end-to-end neurorrhaphy (NRR), is often required. A key pathological feature is Wallerian degeneration (WD) in the distal stump, which, along with slow axonal regeneration, leads to muscle atrophy and poor functional recovery. Polyethylene glycol (PEG)-mediated axonal fusion has emerged as a promising strategy to bypass WD by rapidly reconnecting severed axons and restoring conduction. Methylene blue (MB) has previously enhanced PEG-fusion outcomes. This study investigated whether TEMPOL (TMP), a potent antioxidant, could further improve PEG-fusion-mediated repair following sciatic nerve transection in rats. Adult female Lewis rats underwent unilateral sciatic nerve transection and were randomly assigned to one of three groups: end-to-end neurorrhaphy (NRR), MB-PEG-fusion (MB-fusion), or TEMPOL-PEG-fusion (TMP-fusion). Functional recovery was assessed for 8 weeks using CatWalk gait analysis (Peroneal Functional Index, PFI). Electrophysiological recordings (CMAPs) were obtained at baseline, immediately post-repair, and at 8 weeks. Histological and immunofluorescence analyses (neurofilament, S100, synaptophysin, GFAP, Iba-1) were performed to evaluate axonal integrity, Schwann cell activity, synaptic coverage, and glial response. TMP-fusion significantly improved motor recovery compared to MB-fusion and NRR. Animals treated with TMP-fusion demonstrated superior sensorimotor function by week 8 (PFI; p = 0.0232, step sequence; p < 0.05), enhanced nerve conduction (CMAPs amplitude, p < 0.05), preserved axonal morphology (NF; p < 0.0001, S100, p = 0.0004), and reduced glial activation (Iba-1, p = 0.0035; GFAP, p = 0.001) compared to NRR. Synaptic integrity in the spinal cord was better maintained in the TMP-fusion group, indicating a more complete restoration of neuromuscular connectivity (p < 0.0001; p = 0.0003). TEMPOL-PEG-fusion significantly enhances structural and functional recovery after neurotmesis, outperforming current gold-standard techniques. These results support TMP-fusion as a promising strategy for peripheral nerve repair.

Currently, no effective treatment exists for injuries at the interface between the CNS/PNS, largely due to their complex pathophysiology and the limited efficacy of single‐target therapies. To address this challenge, we investigated a novel combinatorial therapeutic strategy integrating surgical VRR with fibrin sealant biopolymer (FSB) and DMT in a rat model of ventral root avulsion VRA. DMT was extracted from Mimosa tenuiflora roots and structurally characterized using standard analytical methods. Adult female Lewis rats underwent unilateral L4‐L6 VRA and received daily DMT treatment (1, 2.5, or 5 mg/kg; i.p) for 2 weeks to determine the optimal therapeutic dose. Subsequently, the identified optimal DMT dose was combined with VRR, and animals were evaluated 2 weeks post‐injury. Outcome measures encompassed quantitative assessments of neuronal survival, glial reactivity, synaptic preservation, and differential gene expression of neurotrophic factors (GDNF, FGF‐2, VGF‐A) and anti‐apoptotic genes (Bcl‐2, Bcl‐XL). Extracted DMT met all structural and analytical criteria for experimental use. Proximal axotomy led to substantial MN loss (78%), accompanied by pronounced glial reactivity and synaptic detachment. DMT at 1 mg/kg yielded the strongest neuroprotective profile, significantly enhancing MN survival, reducing glial reactivity, and preserving pre‐synaptic boutons. Notably, these effects were further potentiated when DMT treatment was combined with VRR. Moreover, the combined VRR + DMT therapy significantly upregulated GDNF expression, indicating a synergistic effect on neurotrophic support. Overall, our findings suggest that DMT is a promising neuroprotective agent for treating MN degeneration following CNS/PNS interface injuries, particularly when integrated into a combinatorial therapeutic strategy. image