Engineering a 3D microfluidic platform to model neuroimmune interactions in periodontal inflammation

Neuroimmune interactions play a critical role in the progression of periodontal inflammatory diseases such as apical periodontitis (AP), highlighting the need for in vitro systems capable of capturing their multicellular complexity within physiologically relevant microenvironments. Here, we present a novel 3D tri-culture microfluidic platform (3TMP) that integrates human periodontal ligament cells (PDLCs) and macrophages (MQs) in collagen 1 matrix, and neuron-like cells (NLCs) in Matrigel to investigate coupled immune, stromal, and neuronal signaling. Lipopolysaccharide (LPS) stimulation induced temporally distinct inflammatory responses within the device. These responses were notably shaped by the presence of NLCs, underscoring the contribution of bidirectional neuroimmune crosstalk. At 3 h, NLCs were associated with reduced CD80/CD206, STAT1/STAT3, TNF-α, IL-1β, IL-8, and substance P (SP), alongside elevated prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP), indicating an early attenuated inflammatory profile. By 24 h, this shifted towards increased STAT1/STAT6, TNF-α, IL-6, and IL-8, with further elevations in PGE2 accompanied by increased CGRP, IL-10, and TGF-β1, consistent with sustained inflammatory signaling alongside emerging counter-regulatory responses. At the neuronal level, TRP channel dynamics revealed reduced TRPV1 expression despite elevated PGE2, consistent with receptor desensitization, alongside increased TRPA1 expression in the context of inflammatory cytokine signaling. Validation using a mouse model of AP confirmed that cytokine profiles, neuropeptide expression, and TRP channel patterns observed in the 3TMP reproduce key features of in vivo disease. This 3TMP platform provides a human cell-based, physiologically relevant platform for investigating neuroimmune mechanisms and evaluating therapeutic responses in periodontal inflammation.