The effect of the PDGF, vitamin D and PTH on osteoblasts derived from patients on chronic bisphosphonate therapy
Myneni Venkatasatya, Srinivas Rao
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Objectives. Bisphosphonates consist of a family of pyrophosphate analogues, which are potent inhibitors of bone resorption that are currently being used pharmacologically to treat metastatic bone diseases. Recent evidence shows that dental extractions or any kind of trauma in the oral cavity of cancer patients who are chronically using bisphosphonates can lead to "osteonecrosis" of the jaws. In patients with this osteonecrosis, instead of normal healing of bone after trauma, most of the bone becomes necrotic and the wound is not healed. There is a great deal of swelling and pain around the site of injury. Therefore, the purpose of this study was to design therapeutic approaches for the prevention and/or treatment of oral osteonecrosis focusing on 1,25 Vitamin D, PDGF (platelet derived growth factor), and Parathyroid Hormone (PTH) that have all been shown to act as natural wound healing agents on bone. This study was designed to determine whether the remaining viable cells present in an osteonecrotic jaw bone site are responsive to 1,25 Vitamin D, PDGF and/or PTH. Methods. Osteoblastic cells from a diagnosed necrotic alveolar bone specimen obtained with consent from a Multiple Myeloma patient A, Breast cancer Patient B and a non necrotic sample from patient C on chronic bisphosphonates therapy (Zometa®) were successfully cultured. The cells were seeded into six well plates and were exposed to different concentrations of 1,25 Vitamin D, PDGF, or PTH at different time points. The osteoblastic cell differentiation marker, alkaline phosphatase activity, was assayed using biochemical spectrometric analyses. Cell viability was assessed with a MTT assay which measures mitochondrial activity, and cell proliferation with a tritiated thymidine assay. The data were analyzed by ANOVA with comparisons between control and 1,25 Vitamin D, PDGF and PTH treated groups. Results. Our results derived from the experiments conducted on osteoblasts cultured from a necrotic sample (patient A) showed that during the 24 hours incubation PDGF (10 -8 M) significantly increased the ALP levels compare to their control. This PDGF effect was not sustained during the 48 hours incubation, however, 1,25 Vitamin D did result in a significantly increased ALP activity compare to their control during the same time period. During the 24, 48, 72 hours incubation periods with 1,25 Vitamin D at different concentrations, 10 -6 M, 10 -7 M, and 10 -8 M, each significantly increased ALP activity at 24 and 48 hrs compare to their respective controls. Both the bisphosphonate, Etidronate, and the mix of Etidronate and 1,25 Vitamin D significantly increased ALP activity compare to their controls at during 24 and 48 hours incubation. The results of the experiments conducted on osteoblasts derived from non necrotic sample (patient C) indicate, during the short term (1 hour) incubation, with PTH at concentrations of (10 -8 M, 10 -10 M) ALP activity was significantly increased. Long term (48 hrs) treatment with the same concentrations of PTH tended to produce decreases, although not statistically significant, in this parameter. In the measure of cell proliferation, at 48 hours incubation, only PDGF at a concentration of 10 -8 M, produced a significant increase compare to its control, with 1,25 Vitamin D (10 -8 ) and PTH (10 -8 M, 10 -10 M) producing no effects. However, using the MTT assay to measure cell viability, short term (1hr) incubations with PTH (10 -8 M) produced a significant effect. Conclusions. These studies with osteoblastic cells grown out of necrotic alveolar bone from a multiple myeloma patient A and non necrotic sample of patient C on long term bisphosphonate therapy strongly suggest that the viable cells from the bone are responsive to agents such as PTH, PDGF and 1,25 Vitamin D with changes in alkaline phosphatase activity, proliferation and viability suggestive of normal osteoblastic responses. These experiments demonstrate a rationale for further studies to test the clinical effects of 1,25 Vitamin D, PDGF and/or PTH to restore bone loss in patients suffering from bisphosphonate induced osteonecrosis.