Monocyte Chemotatic Protein-1 Induces Hydrolytic Activity in Pulp Fibroblasts

Previous studies showed that monocyte chemotactic protein-1 (MCP-1) was induced in monocyte derived macrophage cells (U937 cells) by triethylene glycol dimethacrylate (TEGDMA). In turn, MCP-1 induced hydrolase activity in human gingival fibroblasts (HGFs) lysates and conditioned media. Objective: The aim of this study was to evaluate the effects of MCP-1 and TEGDMA on the hydrolase activity of human pulp fibroblasts (HPFs) and compare these results to those of HGFs from the previous studies. Methods: HPFs were exposed to 1.25 mM of TEGDMA for 3 hours with or without prior treatment of MCP-1 (10 nM) for 24 hours. Hydrolase activity was measured using a spectrophotometric substrate, p-nitrophenol butyrate. The product of this substrate after cleavage by a hydrolytic enzyme is p-nitrophenyl. Cell media and lysates were normalized for protein concentration. The assays contained 1 µg protein from either cell media or cell lysates and 20 µL (200 ìM) substrate in phosphate buffered saline, pH 7.5, at 25°C in a final volume of 1 mL. Readings at 400 nm, the wavelength at which p-nitro phenol absorbs, were taken every 5 minutes for 1 hour. Results: The relative absorbance that represents hydrolase activity from the cell media exposed to MCP-1 for 24 hours was 0.95 ± 0.08 at the one hour time-point. The relative absorbance representing the hydrolase activity of the control cell media was 0.39 ± 0.02 at the one hour time point. Conclusions: These results indicate that MCP-1 does increase hydrolase activity expressed from the HPFs after a 24 hour exposure time. These results are similar to those seen with HGFs in the previous studies. A hydrolase enzyme released near composite restorations could lead to breakdown of the matrix and resin failure.