RESUMEN
Following peripheral nerve injury (PNI), inflammatory cues impede repair. We have previously demonstrated that spinal cord matrix (SCM) proteins and hyaluronic acid (HA) nanofibers mitigate chondroitin sulfate proteoglycan (CSPG) inhibition and promote growth in peripheral neurons. In this study, we evaluated the effects of a characteristic CSPG, chondroitin sulfate A (CSA), SCM, and HA fibers on macrophages and Schwann cells (SCs). We hypothesized that our cues would accelerate the macrophages' return to rest following classical activation (M1/pro-inflammatory) with lipopolysaccharide (LPS; 1⯵g/mL) and would accelerate the transformation of SCs from an immature state following injury to a mature/pro-myelinating phenotype. LPS stimulation of the macrophages caused upregulation of inducible nitric oxide synthase (iNOS; M1 gene) and led to significantly increased cell area and decreased circularity. However, the SCM and HA nanofibers mitigated this effect, significantly reducing iNOS expression. SCs on the fibers had significantly reduced area and increased elongation. These morphological changes may have polarized the cells leading to decreased GFAP (immature gene) and increased Oct6 and Krox 20 (promyelin genes) expression. Antibody arrays were used to measure relative levels of inflammatory cytokines released by the cells. The arrays confirmed that anti-inflammatory cytokines are released from the cells when cultured with our biomaterial cues and helped identify targets for future investigation including vascular endothelial growth factor (VEGF), interleukin (IL)-10, monocyte colony stimulating factor (M-CSF) from the macrophages, Agrin, ciliary neurotrophic factor (CNTF), tissue inhibitor metalloproteinases (TIMPs)-1 from SCs, and IL-2 from both cell types. In conclusion, these results suggest that our biomaterial cues have pro-regenerative effects on both cell types and if combined may trigger cells toward regenerative programs.
Asunto(s)
Macrófagos/metabolismo , Regeneración Nerviosa/fisiología , Células de Schwann/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Macrófagos/patología , Ratones , ARN Mensajero/metabolismo , Ratas , Células de Schwann/patología , Nervio Ciático/metabolismo , Nervio Ciático/patología , Médula Espinal/metabolismo , PorcinosRESUMEN
Many current peripheral nerve repair strategies focus on delivering positive, growth promoting cues (e.g. extracellular matrix, ECM) while eliminating negative, growth inhibiting cues (e.g. chondroitin sulfate proteoglycans, CSPGs) at the injury site. We hypothesized that recapitulating the positive and negative cues of the peripheral nerve injury microenvironment would improve regeneration. First, we tested the effects of a characteristic CSPG, chondroitin sulfate A (CSA) on neurite dynamics of dissociated chick embryo dorsal root ganglion (DRG) neurons using time lapse video microscopy. DRG growth was recorded on different adhesive substrates, including a novel, porcine-derived spinal cord matrix (SCM). The SCM significantly increased frequency of neurite extension coordinated by a significant reduction in the neurites' time spent stalled. The SCM also mitigated inhibitory effects of CSA, producing longer neurites than the controls without CSA treatment. Next we aimed to elucidate receptors involved in mediating this behavior by testing the ability of CSA to upregulate cell-substrate binding receptors using flow cytometry. Our results showed a significant increase in syndecan-3 receptor expression in neurons treated with CSA. Furthermore, syndecans would most likely bind to the sulfated glycosaminoglycans measured in the SCM. Finally, we evaluated neurite growth on biomaterial scaffolds featuring CSA and SCM cues. Our results showed significantly increased neurite outgrowth on electrospun hyaluronic acid fibers with SCM and low levels of CSA. Higher incorporation of CSA maintained its inhibitory properties. Future work will evaluate coupling CSPGs with growth-permissive ECM to assess the combined effect on neurite outgrowth.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuritas/fisiología , Animales , Materiales Biocompatibles/química , Microambiente Celular/fisiología , Embrión de Pollo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Sulfatos de Condroitina/fisiología , Matriz Extracelular/fisiología , Ganglios Espinales/citología , Ensayo de Materiales , Nanofibras/química , Neuritas/ultraestructura , Traumatismos de los Nervios Periféricos/fisiopatología , Traumatismos de los Nervios Periféricos/terapia , Médula Espinal/fisiología , Porcinos , Sindecano-3/fisiología , Imagen de Lapso de Tiempo , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Directed cell migration is particularly important in neural tissue engineering, where the goal is to direct neurons and support cells across injured nerve gaps. Investigation of the gradients present in the body during development provides an approach to guiding cells in peripheral and central nervous system tissue, but many different types of gradients and patterns can accomplish directed migration. The focus of this review is to describe current research paradigms in neural tissue gradients and review their effectiveness for directed migration. The review explores directed migration achieved through the use of chemical, adhesive, mechanical, topographical, and electrical types of gradients. Few studies investigate combined gradients, though it is known that a combination of therapies is necessary for reconnection of neuronal circuitry. To date, there has been no systematic review of gradient approaches to neural tissue engineering. By looking at effectiveness of various scaffold cue presentation and methods to combine these strategies, the potential for nerve repair is increased.