Mineral-Binding Peptide Inhibits Ectopic Mineralization Secondary to Bone Morphogenetic Protein Stimulation

Abstract

Bone morphogenetic proteins (BMPs) are widely recognized for their therapeutic efficacy in bone regeneration, but one side effect of these therapies is ectopic mineralization. Previous work identified a mineral-binding peptide (pVTK, VTKHNLQI(pS)Q(pS)Y; where pS denotes a phosphoserine) with the ability to inhibit mineralization in osteoblasts. This study investigated the application of pVTK for inhibiting ectopic mineralization secondary to BMP delivery in vitro and in vivo. It was hypothesized that a mineral binding peptide could be delivered alongside BMP to limit unwanted mineralization without limiting the pro-osteogenic effects of the BMP signaling pathway. In vitro, pVTK reduced BMP-stimulated mineral deposition in an osteoblast cell line, as determined by a significant reduction in extracellular matrix calcium deposition with > 300 μM pVTK (p < 0.0001) (at 50 ng/mL BMP2). Importantly, pVTK inhibited mineral deposition without competing with the BMP ligand or diminishing the osteogenic phenotype of the cells in response to BMP stimulation, as demonstrated by no changes in intracellular/extracellular osteogenic protein levels with addition of pVTK. In vivo, pVTK reduced ectopic mineralization of BMP-loaded subcutaneous implants by 92% (p = 0.0101) compared to PBS-treated controls. In an acellular model of spontaneous mineralization, pVTK disrupted mineral deposition and reduced crystallinity and crystal organization (as measured via Raman spectroscopy), demonstrating that pVTK is not solely reliant on cell mechanisms for inhibiting mineralization. These findings support the use of a mineral binding peptide for controlling ectopic mineralization secondary to BMP therapies without interfering with the BMP osteogenic pathway, which is necessary for a regenerative effect.