VPA/PLGA microfibers produced by coaxial electrospinning for the treatment of central nervous system injury.
ABSTRACT: The central nervous system shows limited regenerative capacity after injury. Spinal cord injury (SCI) is a devastating traumatic injury resulting in loss of sensory, motor, and autonomic function distal from the level of injury. An appropriate combination of biomaterials and bioactive substances is currently thought to be a promising approach to treat this condition. Systemic administration of valproic acid (VPA) has been previously shown to promote functional recovery in animal models of SCI. In this study, VPA was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microfibers by the coaxial electrospinning technique. Fibers showed continuous and cylindrical morphology, randomly oriented fibers, and compatible morphological and mechanical characteristics for application in SCI. Drug-release analysis indicated a rapid release of VPA during the first day of the in vitro test. The coaxial fibers containing VPA supported adhesion, viability, and proliferation of PC12 cells. In addition, the VPA/PLGA microfibers induced the reduction of PC12 cell viability, as has already been described in the literature. The biomaterials were implanted in rats after SCI. The groups that received the implants did not show increased functional recovery or tissue regeneration compared to the control. These results indicated the cytocompatibility of the VPA/PLGA core-shell microfibers and that it may be a promising approach to treat SCI when combined with other strategies.
Project description:Secondary damage is a critical determinant of the functional outcome in patients with spinal cord injury (SCI), and involves multiple mechanisms of which the most important is the loss of nerve cells mediated by multiple factors. Autophagy can result in cell death, and plays a key role in the development of SCI. It has been recognized that valproic acid (VPA) is neuroprotective in certain experimental animal models, however, the levels of autophagic changes in the process of neuroprotection by VPA treatment following SCI are still unknown. In the present study, we determined the extent of autophagy after VPA treatment in a rat model of SCI. We found that both the mRNA and protein levels of Beclin-1 and LC3 were significantly increased at 1, 2, and 6 h after SCI and peaked at 2 h; however, Western blot showed that autophagy was markedly decreased by VPA treatment at 2 h post-injury. Besides, post-SCI treatment with VPA improved the Basso-Beattie-Bresnahan scale, increased the number of ventral horn motoneurons, and reduced myelin sheath damage compared with vehicle-treated animals at 42 days after SCI. Together, our results demonstrated the characteristics of autophagy expression following SCI, and found that VPA reduced autophagy and enhanced motor function.
Project description:This study reports a facile method for the fabrication of aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) fibers and tubes based on electrospinning and oxidative chemical polymerization. Discrete PEDOT nano- and microfibers and nano- and microtubes are difficult to fabricate quickly and reproducibly. We employed poly(lactide-co-glycolide) (PLGA) polymers that were loaded with polymerizable 3,4-ethylene dioxythiophene (EDOT) monomer to create aligned nanofiber assemblies using a rotating glass mandrel during electrospinning. The EDOT monomer/PLGA polymer blends were then polymerized by exposure to an oxidative catalyst (FeCl3). PEDOT was polymerized by continuously dripping a FeCl3 solution onto the glass rod during electrospinning. The resulting PEDOT fibers were conductive, aligned and discrete. Fiber bundles could be easily produced in lengths of several centimeters. The PEDOT sheath/PLGA core fibers were immersed in chloroform to remove the PLGA and any residual EDOT resulting in hollow PEDOT tubes. This approach made it possible to easily generate large areas of aligned PEDOT fibers/tubes. The structure and properties of the aligned assemblies were measured using optical microscopy, electron microscopy, Raman spectroscopy, thermal gravimetric analysis, and DC conductivity measurements. We also demonstrated that the aligned PEDOT sheath/PLGA core fiber assemblies could be used in supporting and directing the extension of dorsal root ganglia (DRG) neurons in vitro.
Project description:Since the first work by Laurencin and colleagues on the development of polymeric electrospinning for biomedical purposes, the use of electrospinning technology has found broad applications in such areas of tissue regeneration and drug delivery. More recently, coaxial electrospinning has emerged as an important technique to develop scaffolds for regenerative engineering incorporated with drug(s). However, the addition of a softer core layer leads to a reduction in mechanical properties. Here, novel robust tripolymeric triaxially electrospun fibrous scaffolds were developed with a polycaprolactone (PCL) (core layer), a 50:50 poly (lactic-co-glycolic acid) (PLGA) (sheath layer) and a gelatin (intermediate layer) with a dual drug delivery capability was developed through modified electrospinning. A sharp increase in elastic modulus after the incorporation of PCL in the core of the triaxial fibers in comparison with uniaxial PLGA (50:50) and coaxial PLGA (50:50) (sheath)-gelatin (core) fibers was observed. Thermal analysis of the fibrous scaffolds revealed an interaction between the core-intermediate and sheath-intermediate layers of the triaxial fibers contributing to the higher tensile modulus. A simultaneous dual release of model small molecule Rhodamine B (RhB) and model protein Fluorescein isothiocynate (FITC) Bovine Serum Albumin (BSA) conjugate incorporated in the sheath and intermediate layers of triaxial fibers was achieved. The tripolymeric, triaxial electrospun systems were seen to be ideal for the support of mesenchymal stem cell growth, as shrinkage of fibers normally found with conventional electrospun systems was minimized. These tripolymeric triaxial electrospun fibers that are biomechanically competent, biocompatible, and capable of dual drug release are designed for regenerative engineering and drug delivery applications.
Project description:Histone deacetylases (HDAC) catalyze N-terminal deacetylation of lysine-residues on histones and multiple nuclear and cytoplasmic proteins. In various animal models, such as trauma/hemorrhagic shock, ischemic stroke or myocardial infarction, HDAC inhibitor (HDACi) application is cyto- and organoprotective and promotes survival. HDACi reduce stress signaling, cell death and inflammation. Hepatic ischemia-reperfusion (I/R) injury during major liver resection or transplantation increases morbidity and mortality. Assuming protective properties, the aim of this study was to investigate the effect of the HDACi VPA and SAHA on warm hepatic I/R.Male Wistar-Kyoto rats (age: 6-8 weeks) were randomized to VPA, SAHA, vehicle control (pre-) treatment or sham-groups and underwent partial no-flow liver ischemia for 90 minutes with subsequent reperfusion for 6, 12, 24 and 60 hours. Injury and regeneration was quantified by serum AST and ALT levels, by macroscopic aspect and (immuno-) histology. HDACi treatment efficiency, impact on MAPK/SAPK-activation and Hippo-YAP signaling was determined by Western blot.Treatment with HDACi significantly enhanced hyperacetylation of Histone H3-K9 during I/R, indicative of adequate treatment efficiency. Liver injury, as measured by macroscopic aspect, serum transaminases and histology, was delayed, but not alleviated in VPA and SAHA treated animals. Importantly, tissue destruction was significantly more pronounced with VPA. SAPK-activation (p38 and JNK) was reduced by VPA and SAHA in the early (6h) reperfusion phase, but augmented later on (JNK, 24h). Regeneration appeared enhanced in SAHA and VPA treated animals and was dependent on Hippo-YAP signaling.VPA and SAHA delay warm hepatic I/R injury at least in part through modulation of SAPK-activation. However, these HDACi fail to exert organoprotective effects, in this setting. For VPA, belated damage is even aggravated.
Project description:In previous works, we reported the fabrication of cotton-wool-like composites consisting of siloxane-doped vaterite and poly(l-lactic acid) (SiVPCs). Various irregularly shaped bone voids can be filled with the composite, which effectively supplies calcium and silicate ions, enhancing the bone formation by stimulating the cells. The composites, however, were brittle and showed an initial burst release of ions. In the present work, to improve the mechanical flexibility and ion release, the composite fiber was coated with a soft, thin layer consisting of poly(d,l-lactic-co-glycolic acid) (PLGA). A coaxial electrospinning technique was used to prepare a cotton-wool-like material comprising "core-shell"-type fibers with a diameter of ~12 µm. The fibers, which consisted of SiVPC coated with a ~2-µm-thick PLGA layer, were mechanically flexible; even under a uniaxial compressive load of 1.5 kPa, the cotton-wool-like material did not exhibit fracture of the fibers and, after removing the load, showed a ~60% recovery. In Tris buffer solution, the initial burst release of calcium and silicate ions from the "core-shell"-type fibers was effectively controlled, and the ions were slowly released after one day. Thus, the mechanical flexibility and ion-release behavior of the composites were drastically improved by the thin PLGA coating.
Project description:several genes are being regulated after injury at various time points and after VPA treatment. Overall design: Examination of uninjured, 12hpi, 4dpi and VPA treated 4dpi retinae with duplicate samples.
Project description:Three-dimensional (3D) bioprinting of living structures with cell-laden biomaterials has been achieved in vitro, however, some cell-cell interactions are limited by the existing hydrogel. To better mimic tumor microenvironment, self-assembled multicellular heterogeneous brain tumor fibers have been fabricated by a custom-made coaxial extrusion 3D bioprinting system, with high viability, proliferative activity and efficient tumor-stromal interactions. Therein, in order to further verify the sufficient interactions between tumor cells and stroma MSCs, CRE-LOXP switch gene system which contained GSCs transfected with "LOXP-STOP-LOXP-RFP" genes and MSCs transfected with "CRE recombinase" gene was used. Results showed that tumor-stroma cells interacted with each other and fused, the transcription of RFP was higher than that of 2D culture model and control group with cells mixed directly into alginate, respectively. RFP expression was observed only in the cell fibers but not in the control group under confocal microscope. In conclusion, coaxial 3D bioprinted multicellular self-assembled heterogeneous tumor tissue-like fibers provided preferable 3D models for studying tumor microenvironment in vitro, especially for tumor-stromal interactions.
Project description:Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.
Project description:BACKGROUND:The primary phase of traumatic spinal cord injury (SCI) starts by a complex local inflammatory reaction such as secretion of pro-inflammatory cytokines from microglia and injured cells that substantially contribute to exacerbating pathogenic events in secondary phase. Valproic acid (VPA) is a histone deacetylase inhibitor. Acetylation of histones is critical to cellular inflammatory and repair processes. METHODS:In this study, rats were randomly assigned to five experimental groups (laminectomy, untreated, and three VPA-treated groups). For SCI, severe contusion was used. In treated groups, VPA was administered intraperitoneally at doses of 100, 200 and 400 mg/kg daily three hours after injury for 7 days. To compare locomotor improvement among experimental groups, behavioral assessments were performed by the Basso, Beattie and Bresnahan (BBB) rating scale. The expression of neurotrophins was evaluated by RT-PCR and real-time PCR. RESULTS:VPA administration increased regional brain-derived neurotrophic factor and glial cell-derived neurotrophic factor mRNA levels. Local inflammation and the expression of the lysosomal marker ED1 by activated macrophages/microglial cells were reduced by VPA and immunoreactivity of acetylated histone and microtubule-associated protein were increased. CONCLUSION:The results showed a reduction in the development of secondary damage in rat spinal cord trauma with an improvement in the open field test (BBB scale) with rapid recovery.