Placental Allografts Provide Multi-Modal Immunomodulation for Improved Peripheral Nerve Regeneration
Victoria Stefanelli, PhD, Sita Damaraju, PhD, Sunil Saini, PhD and Ankur Gandhi, PhD, Integra LifeSciences, Princeton, NJ
Favorable immunomodulation is essential for facilitating quick and complete peripheral nerve repair. Lack of IL-4 and infiltrating T cells have been cited as major deficits in the ability of acellular nerve allografts to satisfactorily bridge large defects. Further, strategies to pointedly modulate the local immune environment such as through improving the presence of M2 pro-regenerative macrophages or T regulatory cells have demonstrated significant enhancements in permissiveness of nerve conduits. Amniotic membrane wraps have previously demonstrated significant benefits to peripheral nerve regeneration across multiple pre-clinical studies. Here, we comprehensively evaluate the ability of dehydrated tri-layer placental allograft membranes (TPAM) to favorably modulate immune functions known to benefit peripheral nerve regeneration in order to better elucidate their utility as nerve wraps.
Materials & Methods:
The immunomodulatory capability of TPAM was investigated in a four-pronged manner considering M1/M2 macrophage polarization states, T regulatory cell differentiation, Th1/Th2 helper T cell subsets, and overall inflammatory cytokine presence. The broad anti-inflammatory functionality of TPAM was evaluated via multiplex cytokine analysis following PBMC activation with lipopolysaccharide in the presence of TPAM extracts. Multiplex analysis was also used in assessment of macrophage polarization states through measurement of CCL22, CCL18, PDGF, IL-13, TNF-alpha, IFN-gamma, IL-6, and RANTES following exposure to TPAM. Two distinct flow cytometry panels were employed to assess relevant lymphocyte characteristics following phytohemagglutinin (PHA) stimulation in the presence of TPAM extract; these included lymphocyte proliferation, T regulatory cell (CD25+/FoxP3+) differentiation, and ratios of Th1(IL-2+) versus Th2 (IL-4+) T helper cell subsets.
Exposing naïve macrophages to TPAM resulted in an M1:M2 ratio of 0.39, strongly in preference of pro-regenerative M2 markers. Cytokine analysis indicates that TPAM extracts possess a significant ability to reduce secretion of pro-inflammatory molecules including TNF-alpha, IL-1beta, IL-10, MIP-1a, and IL-6 by over 90% in PBMC compared to controls (n=4 per group; p<0.05). T regulatory cell presence increased in a dose-dependent manner up to over 3-fold in the presence of TPAM extract (see figure) compared to non-treated (NT) PHA-activated PBMCs. IL-4-positive T-helper cells exposed to TPAM extracts significantly expanded while concurrently decreasing IL-2 production, thus demonstrating a strong Th2 phenotype preference.
From an immune stand-point, placental allografts are more than just passive low-immunogenicity constructs; they directly modulate the immune response in a multifaceted approach. This immunomodulation includes suppression of inflammation and promotion of pro-regenerative macrophage and T cell subsets that can actively contribute to peripheral nerve regeneration.
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