Quantitative determination of matrix Gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells

Abstract

Background and objectiveMatrix Gla protein (MGP) has been recognized as a potent calcification inhibitor and a regulator for bone morphogenetic protein-2 (BMP-2). The periodontal ligament (PDL) is a non-mineralized connective tissue located between two mineralized tissues, the cementum and the alveolar bone. However, the mechanism by which PDL prevents mineralization has yet to be defined. This study aims to examine the expression pattern of MGP and BMP-2 during human periodontal ligament cells (hPDLCs) osteogenic differentiation in vitro, preliminarily exploring their roles in this process. Materials and methodshPDLCs were obtained and cultured in mineralizing medium. The expression of MGP and BMP-2 was confirmed by RT-PCR and immunofluorescence staining. In the process of osteogenic induction, alkaline phosphatase (ALP) activity, extracelluar calcium deposition, and mineralized nodules were measured. Quantitative real-time RT-PCR was performed to evaluate mRNA expression of MGP, BMP-2 and osteogenic marker genes, including ALP, bone sialoprotein (BSP), type I collagen (COLI), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). The protein expression of MGP and BMP-2 was analyzed by western blotting. ResultsCo-localization of MGP and BMP-2 was visualized in hPDLCs. After osteogenic induction, ALP activity, calcium deposition, mineralized nodules, and osteogenic marker genes were significantly up-regulated. mRNA expression of MGP and BMP-2 generally decreased, although MGP mRNA increased on day 14 and 21 compared with the control, and protein expression of MGP and BMP-2 was down-regulated. ConclusionOur results indicate that MGP might regulate hPDLCs osteogenic differentiation which might keep a potential relationship with BMP-2 in this process.