Biomineralogical Signatures of Pathlogical Mineralization

Pathological calcification is a wide-spread phenomenon in the human body, in which calcium minerals form in soft tissues and are found in both healthy and diseased tissues. One example is calcific aortic valve disease (CAVD), which affects roughly 26% of people over the age of 65. In CAVD, mineralized lesions form in the leaflets of the aortic valve. Progressive mineralization causes valve leaflets to stiffen and impairs their ability to regulate blood flow into the aorta, leading to an increased risk of heart failure. The goal of this work is to characterize the “biomineralogical signatures” of a range of disease conditions and develop an <i>in vitro</i> model that recapitulates key aspects of the early stages of CAVD that precede the onset of symptoms. We hypothesize that the formation and growth of calcific nodules in the aortic valve is modulated by interactions of valve endothelial and valve interstitial cells with calcium phosphate nanoparticles in the extracellular matrix. From characterization of excised human heart valve leaflets, we know that there are multiple types of calcium phosphate nanoparticles present during disease progression. We explore these interactions through tissue engineered constructs mimicking human aortic valve leaflets. The in vitro tissue models of aortic valve leaflets consist of a collagen hydrogel under uniform static stress, seeded with porcine valve interstitial cells (VICs) and a surface layer of porcine valve endothelial cells (VECs). These 3D collagen constructs can also be seeded with synthetic calcium phosphate nanoparticles of varying phase – hydroxyapatite (HAp; Ca₁₀(PO₄)₆(OH)₂) and whitlockite (Wh; (Ca,Mg)₃(PO₄)₂). We have synthesized nanoparticles of HAp and Wh (phase validated by x-ray diffraction and Raman spectroscopy) and incorporated them into the cellularized spring gel constructs to create a 3D in vitro model of calcific aortic valve lesions. Incubation in general (GM) and osteogenic media (OGM) leads to rapid (~1 week) formation of lesions within the constructs; these lesions and their environment are characterized by Raman mapping, immunofluorescence, and histology. Raman mapping of these tissue engineered constructs has revealed that lesion composition and morphology varies with the type of growth media used, and the type of added nanoparticles. Ongoing and future experiments will aim to expand our understanding of the role that mineral plays in driving the disease progression of CAVD, ultimately applying our understanding of calcific lesion development towards strategies for prevention and treatment of CAVD.