Deciphering the Interplay Between Macrophages and Matrix Formation in the Peripheral Nerve

Introduction: : While peripheral nerves possess the innate ability to regenerate across small gap injuries, large gap peripheral nerve injuries (PNI) require surgical intervention. Recently the emergence of biomaterials for PNI seek to improve functional recovery of large-gap injuries. Although these materials may show potential clinical translation when tested in vitro, these materials commonly incite a foreign body response (FBR) when implanted in vivo. This occurs when the body is either unable or inefficient at degrading the implant. The immune system reacts by segregating the implant from the target tissue through the aggregation of macrophages and fibrous capsule formation. Inflammation occurring because of FBR in vivo impedes long term integration of the material and reduces effective recovery of the damaged tissue.

During PNI, the extracellular matrix (ECM) also plays a crucial role in facilitating regeneration through its relationship with resident cells in the tissue. A comprehensive understanding of the changes occurring in the ECM due to mediators produced during inflammation and regeneration holds the potential for improving the long-term outcomes of implantable biomaterials. This work seeks to gain fundamental knowledge on how a neural ECM is modulated in response to immune stimuli and how cell-ECM interactions affect the regenerative cells of the PNS.

Materials and

Methods: : RAW264.7 macrophages were polarized utilizing LPS/IFNY to produce M1 and IL-4 to produce M2 phenotypes. The phenotypic states were confirmed using RT-qPCR for iNOS (M1) and Arginase-1 (M2). After 24 hours with stimulating media, standard growth media was added, and the cells were left to secrete phenotype specific cytokines and growth factors. Conditioned media was harvested after 24 hours with each corresponding phenotype, terminally filtered and stored at –20°C. Cytokine concentration in conditioned media was measured using ELISAs for TNF-A, IL-6 and IL-10.

Rat perineurial fibroblasts were cultured for 14 days and decellularized using hypertonic solutions and detergents. The decellularized ECM (dECM) was stained with anti-fibronectin and DAPI to validate decellularization and confirm absence of nuclear material. Decellularized ECM was solubilized, quantified using a BCA assay and run through nano LC-MS/MS. For genetic analysis of RPF, cells were cultured using a 50/50 mixture of conditioned media and RPF growth medium for 14 days with changing media every other day. On the final day, RNA from the cells was purified and RT-qPCR for genes associated with the ECM were analyzed.

RC96 Schwann cells were seeded on dECM and cultured for a minimum of three days. Time-lapse videos and images were used to measure cell alignment, cell aspect ratio and cellular migration using NIS-Elements AR software. In addition to the physical evaluation a molecular evaluation was conducted through gene studies using RT-qPCR for c-JUN, p75NTR, GFAP, Krox20 and Krox24.

Results, Conclusions, and Discussions:: RT-qPCR results for iNOS and Arg-1 showed a statistically significant difference regarding gene expression of those not treated (M0), those polarized with LPS/IFNY (M1) and the cells stimulated with IL-4 (M2) (Figure 1A). ELISA experiments showed that there are significant differences in concentrations of TNF-A, IL-10 and IL-6 within the conditioned media for each macrophage phenotype (Figure 1B). After being cultured in macrophage-conditioned media, RPF cells displayed significant differences in their normalized expression for fibronectin (FN1) (Figure 1C). Although the other genes being examined displayed slight differences there was no statistically significant difference between the groups. Phase and fluorescent images showed that there were no remaining nuclei in the decellularized extracellular matrices when stained with DAPI. These results confirm the efficacy of our decellularization procedure (Figure 2). LC-MS/MS analysis showed 858 proteins detected within the soluble and insoluble fractions of the RPF dECM. These proteins consisted of those associated with tight junctions, histone modification, and serine proteases. When cultured on modified dECM Schwann cells appeared more aligned on the dECM grown with M0 and M2 conditioned media while those cultured on M2 dECM showed a more aberrant arrangement.

These results indicate that the presence of inflammation within the peripheral nerve microenvironment changes the composition of the ECM and alters the incidence of Schwann cells expressing the regenerative phenotype. These results demonstrate the importance of considering the immune system and ECM when designing implantable materials. Long term, this work aims to transform the landscape of regenerative medicine by improving the design of biomaterials that can be applied to the peripheral nervous system, and enabling functional recovery for large gap injuries.

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