Immunomodulatory effects of apical papilla cells on periodontal ligament fibroblasts stimulated with Escherichia coli lipopolysaccharide

an in vitro study

Abstract

The role of human Stem Cells from the Apical Papilla (SCAP) in tissue regeneration has been described, but their impact on modulating the apical inflammatory process by other surrounding cell populations, such as periodontal ligament fibroblasts (PLFs), is unclear. Therefore, we investigated the role of SCAP in the activation of PLFs in vitro. Methods  Primary SCAP culture was used to obtain conditioned media (CM). A primary human PLF culture was established and stimulated with increasing concentrations of Escherichia coli lipopolysaccharide (LPS) (0.01, 0.1, and 1 µg/mL). At the 24 h time-point, an MTT viability assay was performed, and interleukin (IL)-6 and chemokine (CC-motif) ligand 2 (CCL2) levels were quantified by enzyme-linked immunosorbent assay. Then, PLFs were stimulated with LPS in the presence of SCAP-CM (1:5 dilution) for cell viability assessment and cytokine detection. The following groups were tested: PLF activated with LPS at concentrations of 0.01 and 1 µg/mL with or without SCAP-CM; a group with PLF stimulated by SCAP-CM alone; and a control group (proliferation medium only). The experiments were conducted in triplicate and sextuplicate. Statistical analyses were performed using analysis of variance followed by Tukey’s post-hoc test, with statistical significance established at 5% (p=0.05). Results  The MTT assay showed no cytotoxicity of LPS or SCAP-CM on PLFs (p>0.05). The production of CCL2 and IL-6 significantly increased in the presence of SCAP-CM regardless of the presence of LPS (p<0.0001). Conclusion  SCAP-CM significantly enhanced the release of proinflammatory cytokines by PLFs in vitro.