The effects of salivary and egg yolk phosphoproteins on bone remodeling using an ex vivo neonatal mouse calvaria bone organ culture system

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact [email protected] (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2014 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 147-167.Previous in vitro and in vivo studies have demonstrated that the phosphorylation state of extracellular matrix (ECM) proteins plays an important role in the modulation of bone mineralization and subsequent homeostasis. Furthermore, several salivary phosphoproteins, including acidic proline-rich proteins, histatins, and statherin, have been shown the ability to bind calcium, interact with hydroxyapatite (HA) surfaces, and promote enamel remineralization. Based upon extensive work carried out using bone ECM phosphoproteins, which are important in the formation and regulation of biomineralization of bone and bone cellular behavioralactivity, we postulate that the most highly phosphorylated protein of egg yolk, phosvitin, will have biological functions that, to date, have not been investigated. In order to evaluate the biological functions of salivary phosphoproteins and phosvitin, this study utilizes a true living ex vivo mouse calvarial organ system to study the effects of these phosphoproteins on bone formation and resorption via the recruitment and differentiation of local progenitor cells into mature osteoclasts and osteoblasts. This model system has been developed to permit studies to be carried out under conditions that dissociates the two bone remodeling stages, e.g. osteoclastic bone resorption or osteoblastic bone formation. Calvaria were cultured for 10 days, following which, histomorphometric and biochemical changes were evaluated. Qualitative assessments of the resorption system were made by staining the vital calvarial bone with the Neutral Red, for osteoclast activity, followed by silver counterstaining to reveal areas of gross resorption. All groups in both the resorption and formation systems were also stained with hemotoxylin and eosin and histological serial sections were examined. Quantification of chemical and biochemical changes were done by measuring relative media calcium changes, as well as tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) enzymatic activity for resorption and formation systems, respectively. Overall, the present study, for the first time, established an exciting new roles for Histatin-1, acidic proline-rich protein-1 (aPRP-1), and phosvitin in bone biology. Importantly, we have shown that the presence of Histatin-1 in the resorption system has the novel ability to enhance osteoclastic bone remodeling events. The major discoveries related to egg yolk phosvitin and aPRP-1 in inhibition of PTH-induced osteoclast differentiation and bone resorption provided novel biological functions of these proteins. These, combined with ground breaking activity of phosvitin in the stimulation of osteoblastic differentiation and new osteoid and bone formation opens new avenues of important scientific investigations with major impact