Presence of Chondroitin Sulfate in Nerve Conduits Promotes Favorable Immunomodulation for Peripheral Nerve Regeneration
Victoria Stefanelli, PhD, Andrew Mesaris, MS, Benjamin Mintz, PhD, Elaina Panas, PhD, Simon Archibald, PhD and Ankur Gandhi, PhD, Integra LifeSciences, Princeton, NJ
Introduction
Chondroitin sulfate (CS), a pervasive component of the nervous system, plays a key role in axonal guidance during embryonic development and nerve regeneration processes, and it is upregulated at sites of neuronal injury. CS is also a component of decellularized allograft nerves (dANs), and it has been demonstrated to significantly promote schwann cell infiltration when added to matrices designed to support peripheral nerve regeneration (PNRM). Elucidation of CS's interactions with cellular players during inflammatory responses is thus important for understanding its role in nerve regeneration. Previous studies have demonstrated CS's ability to reduce neuroinflammation and modulate macrophage polarization, phenomena known to positively influence nerve regeneration. Here, we explore the ability of CS, as well CS-PNRMs to modulate immune responses during nerve regeneration.
Materials & Methods:
Immunofluorescent staining of multiple CS-subtypes and laminin within dAN cross-sections was performed to confirm the presence and localizations of CS relative to major internal nerve structures. Macrophage polarization assays with multiplex analysis of secreted CCL22, CCL18, PDGF, IL-13, TNF-alpha, IFN-gamma, IL-6, and RANTES in the presence of soluble CS or CS-PNRM were employed to determine the ability of CS to promote pro-inflammatory M1 vs pro-regenerative M2 macrophage responses. The effect of CS on inflammation was explored via flow cytometry analysis of human PBMCs stimulated with phytohemagglutinin in the presence of isolated CS or CS-PNRM and with measurement of secreted pro- and anti-inflammatory cytokines, including IL-2 and IL-4.
Results:
Positive chondroitin-6-sulfate staining was detected predominantly within the fascicles of dANs and co-localized with endoneurial tubes, whereas chondroitin-4-sulfate was localized to the perineurium. Macrophage polarization studies revealed M1:M2 ratios of <1 for both isolated CS and CS-PNRM, both of which were lower than that of collagen-only controls, indicating that CS is the primary factor promoting pro-regenerative M2 macrophage phenotypes. Flow cytometry results indicate an overall reduction of pro-inflammatory IL-2 concurrent with up to a 4-fold increase of cells producing anti-inflammatory IL-4 in the presence of CS or CS-PNRM.
Conclusions:
In this model, CS acting either in isolation or within collagen constructs is capable of reducing local inflammation as well as modulating macrophage responses in favor of regeneration. The robust presence of CS within the fascicles of dANs as well as its positive influence on nerve regeneration in PNRMs indicates that CS modulation of inflammatory processes is an important contributor to the outcome of peripheral nerve regeneration.
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