Musculoskeletal diseases, in particular osteoporosis, are increasingly becoming more prevalent in the U.S. due to the ageing population (Figure1). It is estimated that one-sixth of 300 million people in U.S. suffer from bone disorders or loss. About 10 million of those people above age 50 suffer from osteoporosis. Patients that suffer from osteoporosis have high morbidity and mortality rates. For instance, patients have decreased bone mineral density (BMD), a measurement of bone density that reflects the strength of bone as represented by calcium content. A decrease in BMD typically leads to an increased risk of bone fractures. In particular, hip fractures have an associated 20% mortality rate 1 year after injury among senior citizens 1. Patients that suffer from musculoskeletal diseases and from bone injuries, not associated with disease, account for 130 million hospital visit per year. Not to mention, 245 billion dollars of healthcare expenditure 2. Over that last 30 years, there has been much improvement in the field of bone research and its application to medicine. It has changed the quality of life and prolonged the life expectancy of patients suffering from bone disease. However, many details remain unknown about the underlying mechanism that control bone metabolism, formation, and healing. Furthermore, current effective therapies to combat bone disorders have limitations including unwanted side effects and prohibitive costs. For example, treatment with glucocorticoids which is a known inducer of osteoblastogenesis in vitro has been shown to produce an osteoporotic phenotype in vivo. Recognizing the importance of bone health and its affordability to the public makes the advancement of therapeutic targets work worth doing. Work in this field will eventually lead to the prevention, treatment, and cure for bone disease. A potential therapeutic candidate that maybe involved directly or indirectly with bone formation is secreted phosphoprotein-24 (Spp24). The following research aims to establish an importance and role for Spp24 in bone differentiation. A novel antibody that detects Spp24 which we have developed and characterized, has allowed us to feasibly study the protein. Our results demonstrate localization of Spp24 in different tissue, the processing of the protein during osteoblastogenesis, and have allowed us to conceptualize possible functions based on our data.

Secreted Phosphoprotein-24 (Spp24) was initially isolated and characterized as a component of bovine cortical bone matrix. Subsequent characterization has shown it is multiply phosphorylated and homologous to cystatin and TGF-β receptor type II. Spp24 is a minor component of the serum fetuin mineral complex that binds calcium-phosphate minerals and prevents their deposition. The TGF-β receptor homology domain binds BMP-2 weakly in vitro and enhances BMP-2’s osteogenic effects in vivo. The ability of Spp24 to affect BMP activity suggests an important role for Spp24 as a native, bioactive componentof bone that regulates bone development. Spp24 was highly up-regulated in rat cortical kidneys following a low calcium diet regime. Tissue distribution of both Spp24 protein and RNA showed that while Spp24 accumulates in bone, a majority is produced at distant sites, namely the liver and kidney. Additionally, Spp24 was present in more tissues than previously believed. Spp24 migrates to a number of different molecular weights, suggesting multiple, alternative posttranslational modifications may generate subtly different forms of the protein. Theexpression of Spp24 in the kidney may be regulated to counteract changes in serum mineral levels. Additionally, homology in the Spp24 sequence suggests that it, like other bone and dentine matrix proteins, may interact with mineral as an important influencer of mineral calcification. Utilizing microarray analysis of primary bone marrow-derived mesenchymal stem cells transduced with Spp24 and control viruses we examined changes elicited by the overexpression of Spp24. A change in overall morphology was observed for cellstransduced with the Spp24 similar to changes described in cells undergoing osteoblasticdifferentiation. Nodule formation was also seen in the Spp24 transduced cells. Microarray results showed key markers of osteoblast differentiation, CBFA1/RUNX2 and osterix(OSX), were not up-regulated although there were distinguishable changes in the gene expression profile of mesenchymal stem cells. The cells appeared to be blocked from differentiation into a number of mesenchymal lineages: adipocytes, myocytes andchondrocytes. The changes appeared to prime cells for signals that activate osteoblastdifferentiation by blocking other pathways and altering internal signaling response pathways to those signals. This document was created in Microsoft Word 2003.