Multianalysis of AGEs Associated with Calcification in Cultured RDPCs

Objectives: Pulp stones have been reported in patients with type II diabetes mellitus. We have previously analyzed diffuse calcifications in dental pulp tissues of type II diabetes model rats using immunohistochemistry, genetic engineering and other techniques, revealing associations between pathological calcification in rat dental pulp tissues and interactions between pentosidine, S100A8/A9 protein and receptor for advanced glycation end-products (RAGE). The aim of this study was to elucidate the effects of advanced glycation end-products (AGEs) on the calcification ability of cultured rat dental pulp cells (RDPCs).
Methods: RDPCs were isolated from incisors of 6-week-old male SD rats. Third-passage cells were seeded on well plates. AGEs were prepared with non-enzymatic glycation of type I collagen-coated plates by DL-glyceraldehyde. AGE formation on collagen was detected by high-performance liquid chromatography. RDPCs were cultured on AGE-modified collagen-coated dishes in odontogenic induction medium for 21 days. To investigate the effects of AGEs on RDPC, we performed WST-1 proliferation assay, alkaline phosphatase (ALP) staining, Alizarin-Red staining and RT real-time PCR. Pathological calcification was also observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and analyzed by energy-dispersive X-ray spectroscopy (EDX).
Results: AGEs had no significant effect on RDPC proliferation and ALP activity until day 14, but significantly promoted calcium nodule formation. TEM confirmed the presence of matrix vesicles in contact with RDPCs on the collagen-coated side, and SEM confirmed pericellular calcium deposits. Calcium (Ca) and phosphorus (P) peaks were detected by EDX analysis.
Conclusions: AGEs do not affect RDPC proliferation or differentiation, but do appear to enhance calcification. AGEs have direct effects on calcified matrix formation in differentiated RDPCs. The details of calcification via the AGE/RAGE pathway remain unclear, so further consideration is needed to elucidate target pathways and proteins.