Biomaterial Cues to Direct a Pro-regenerative Phenotype in Macrophages and Schwann Cells

Abstract

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 μg/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.

Keywords: PCR; Schwann cells; chondroitin sulfate; cytokines; extracellular matrix; hyaluronic acid; macrophages; nanofibers.