Pathological calcification is the abnormal deposition of minerals in soft tissues or the premature or excessive calcification of normally mineralizing tissues, and is associated with several conditions such as coronary heart disease, craniosynostosis and fibrodysplasia ossificans progressiva. Treatments are limited in their ability to prevent pathological calcification, thus necessitating the development of alternative therapeutic strategies. The work presented in this dissertation involves the use of a newly discovered phosphopeptide in testing inhibition of mineralization in vitro and treatment of pathological calcification in vivo. The global hypothesis was that phosphorylated apatitespecific peptide, pVTK (VTKHLNQIpSQpSY), will inhibit mineralization by physicochemical and/or cellular pathways involving the TNAP-Enpp1-Ank axis. Further, it was hypothesized that pVTK will prevent premature hypermineralization, suture fusion and abnormal craniofacial morphology in mouse models of craniosynostosis.

Phosphorylation of serine residues resulted in increased pVTK binding to carbonated apatites, as well as dose-dependent inhibition of osteoblast mineralization. Peptide phosphorylation was more important than amino acid sequence order for binding to synthetic apatite substrates, whereas regulation of osteoblast culture mineralization was determined by both serine phosphorylation and sequence order. pVTK was internalized by osteoblasts and following treatment for 10-12 days, downregulation of inhibitors of mineralization (Enpp1, osteopontin and Ank) and upregulation of promoters of mineralization (TNAP, Runx2 and bone sialoprotein) was observed. It was further demonstrated that mineral inhibition was not due to disruption of collagen matrix production or calcium chelation with negatively charged pVTK.

Several murine models of pathological calcification were evaluated as potential candidates to demonstrate peptide-mediated inhibition of calcification. pVTK was delivered to prematurely mineralizing anterior-frontal cranial sutures in a BMP-linked mouse model of craniosynostosis. Treatment with pVTK caused a partial correction of craniofacial defects, but did not inhibit primary osteoblast mineralization in vitro or premature suture fusion in vivo. Additionally, pVTK caused a decrease in bone quality, indicating negative effects of peptide on bone growth or turnover.

Further in vitro and in vivo evaluation of the mechanisms underlying pVTK-mediated inhibition of mineralization will enable the development of a novel class of peptide-based drugs, capable of preventing pathological calcification of tissues and biosprosthetic heart valve implants.