Continous Parathyroid Hormone (PTH) Inhibits Mineralization of MLO-A5 Producing a Dystrophic Mineral in vitro

Objective: It has been postulated that bone loss is due to acceleration of bone resorption over effects on bone formation. PTH has been shown to directly inhibit osteoblast nodule formation in vitro, suggesting a direct effect (Bellows et al, 1986). It is not clear if PTH has effects on the proliferation of osteoblast precursors and/or on the maturation of osteoblasts. In our experiments, we sought to determine whether continuous PTH has an effect on the mineralization process. Methods: We used the MLO-A5 cells that mineralize forming sheets within one week of culture. Time courses and dose responses of PTH treatments were performed. These cells have been shown previously to make bone-like hydroxyapatite by FTIR (Kato et al, 2001). To quantitate mineralization, the cells were stained with Von Kossa., then quantified using Optimas image analysis software. Cells were analyzed using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Collagen type I was assessed using immunofluorescence and an antibody (LF-76) provided by Dr. Fisher. Results: MLO-A5 cells mineralize forming honeycombed structures within one week of culture with the addition b-GPO4 and ascorbic acid. These cells synthesize large amounts of collagen type I, alkaline phosphatase, bone sialoprotein and osteocalcin in the early stages of culture (Kato et al, 2001). Previous microscopic analysis showed that MLO-A5 cells produces an extracellular matrix rich in collagen type I, onto which calcium phosphate is deposited. With the addition of continuous PTH at a concentration of 10-8M to MLO-A5 cells, mineralization decreased significantly. SEM showed clusters of cells and very few mineralized structures shown to be dystrophic by TEM. TEM also showed absence of collagen fibrils. Conclusions: These results suggest that PTH can have a direct effect on the mineralization process, potentially by decreasing the production or assembly of collagen type I. NIHAR46798