Inhibition of Ectopic Mineralization with Mineral-Binding Peptide

Abstract

Ectopic mineralization describes the formation of unwanted and non-physiological mineralization that primarily affects soft tissues and results in pain and dysfunction. Though ectopic mineralization is often one component of a larger disease state (e.g. coronary arterial calcification, kidney stones, calcific tendinitis, heterotopic ossification), intensifying ectopic mineralization is associated with worsening patient outcomes. However, the etiologies of most of these disease states are not fully known, complicating the search for disease-specific therapeutic approaches. Therefore, directly targeting and mitigating ectopic mineralization itself is proposed as an additional therapeutic strategy with broad potential application. The work presented in this thesis investigates a phage display-derived mineral-binding peptide (pVTK, VTKHLNQI(p)SQ(p)SY, where p denotes phosphorylation of the serine residues) for the inhibition of ectopic mineralization. It was hypothesized that pVTK could inhibit ectopic mineralization regardless of disease state and that pVTK physically restricts mineral formation and growth to achieve inhibition. These hypotheses were tested in two disease states involving ectopic mineralization, one involving the off-target effects of an exogenous osteogenic promoter and the other involving downstream effects of a genetically modified osteogenic signaling pathway. BMP2 (bone morphogenetic protein 2) is a major regulator of osteogenic differentiation and is FDA-approved for augmentation of bone regeneration in specific surgical applications. However, due to implant mishandling and off-label usage, there are major side effects, including ectopic mineralization. In this thesis, pVTK was shown to inhibit BMP2-stimulated mineralization in vitro and in vivo. In culture, pVTK did not disrupt osteoblast recognition of exogenous BMP2, nor did pVTK compete with BMP2 for control over osteogenic differentiation. Since BMP2 therapies are designed to aid bone regeneration, it was important to demonstrate that pVTK would not decrease BMP2 efficiency by interfering with BMP2 signaling. In vivo, pVTK reduced ectopic mineral formation by 92%. Further, pVTK disrupted spontaneous mineral deposition in the absence of cells, demonstrating that pVTK does not rely on cell-mediated mineralization mechanisms for inhibition. Since pVTK’s inhibition is not reliant on a specific cellular pathway, pVTK would not be considered disease-specific, and therefore broadly applicable for inhibiting ectopic mineralization. Ectopic mineralization is the hallmark of heterotopic ossification (HO), a disease state involving endochondral ossification of soft tissue, and fibrodysplasia ossificans progressiva (FOP), a genetic disorder in which regulation of the osteogenic BMP pathway is impaired. In this thesis, pVTK was shown to reduce ectopic mineralization by 61% in an animal model of HO that utilizes an activatable genetic mutation inspired by FOP. pVTK treatment also resulted in more fragmented ectopic mineral with no change in overall density, supporting the hypothesis that pVTK acts directly on mineral assembly and formation to achieve inhibition. New micro computed tomography methods for assessing the morphology of ectopic mineral deposits were introduced, demonstrating alternative qualitative methods for evaluating amorphous 3D structures. Altogether, this work demonstrated the potential for pVTK as a broadly applied therapeutic for ectopic mineralization. In addition, controlled release delivery systems were piloted for improving pVTK delivery in each disease state: an injectable collagen-alginate hydrogel for delivery alongside BMP2-loaded implants and PLGA (poly-co-lactic-glycolic acid) particles for minimizing intramuscular injections in HO/FOP. Further, based on pVTK’s high affinity for mineralized substrates and pVTK’s unique ability to inhibit mineralization, pVTK could be applied to mineral-targeted drug delivery and musculoskeletal tissue engineering strategies.