Detection of inflammatory mediators in collected dentinal fluid

Objective: Pro-inflammatory cytokines and chemokines are produced in mucosal epithelium and secretions in response to local microbial infections. Little is known about the presence and concentration of innate immune and inflammatory mediators in dentinal fluid. We hypothesize that they may occur in dentinal and pulp fluids in response to infections. The objective of this study was to evaluate accurate collection of unadulterated dentinal fluid and determine the concentrations of cytokines and chemokines. Method: Freshly extracted sound third molars (n=21) had their roots trimmed up to the pulp horns exposing dentin, followed by 10s acid etching (35% H3PO4) and rinsing for 10s to open dentinal tubules and facilitate outward flow. Then, teeth were placed into a conical tube and centrifuged at 4000 rpm for 30 min at 4oC. Collected dentinal fluid (1-2 μl) was placed in labeled tubes containing 300 µl 0.01 M PBS, pH 7.2 and immediately frozen at -80 C until further analysis. Concentrations of cytokines and chemokines (pg ml-1) were determined using a commercial multiplexed fluorescent bead-based immunoassay (Kit 48-002, Millipore, Billerica, MA) in the Luminex 100 IS Instrument (Luminex, Austin, TX). Results: Dentinal fluid was collected and low concentrations (mean±SD) of cytokines and chemokines were detected as follows: Th1 cytokine IL12p70 (18.2+1.3, range 10.0-12.4); Th2 cytokine IL10 (10.9+0.5, 16.2-21.5); proinflammatory cytokines IL1β (3.5+2.1, range 1.8-13.8), IL6 (20.5+6.7, 16.4-36.0), and TNF-α (8.3+0.9, 5.8-10.4); and chemokine CXCL8/IL8 (18.3+32.3, 6.0-154.0). Conclusion: Unadulterated dentinal fluid can be extracted for determination of basal levels of cytokines and chemokines. It will be possible to differentiate among subjects with basal levels and those that appear to have infection or inflammation. (Supported by Research Seed Grant Program, College of Dentistry, University of Iowa)