Biophysical analysis of dystrophic and osteogenic models of valvular calcification.

The pathogenesis of CAVD involves the deposition of calcium rich nodules on the fibrosa layer of aortic valve leaflets [1–3]. The presence of these structures significantly impedes proper opening and closing of the valve, leading to left ventricular pressure overloading and eventual heart failure [2,4]. Two major types of valvular calcification have been observed in diseased excised tissue: dystrophic and osteogenic [5]. Dystrophic calcification is the predominant form of valvular calcification being found in 83% of diseased valves and is described as an amorphous crystalline material [6]. Osteogenic calcification is present in 13% of valves containing dystrophic calcification and is identified by the presence of osteoid matrix reminiscent of active bone formation [6]. At present, there exists only surgical intervention for CAVD, which although effective, includes a 3% mortality rate and is only utilized at the end stage disease [7,8]. Efforts to describe the mechanisms of valvular calcification may lead to the development of novel pharmacological treatments for CAVD [9–11]. The etiology of CAVD has been extensively studied through the development of valvular calcification in vitro models [5,10,12–22]. These models describe the formation of CNs via aortic valve interstitial cells (AVICs) in unique culture conditions and are thought to mimic the dystrophic and osteogenic calcification found in diseased explanted aortic valve leaflets [5,6]. The CNs generated from each model appear morphologically similar in that they are cellular aggregates but form via distinct mechanisms and have unique features. Dystrophic nodules form on stiff substrates, are characterized by cell death, and involve the differentiation of quiescent AVICs into activated myofibroblasts via inflammatory cytokines such as TGF-β1 [14,15,23,24]. Conversely, osteogenic nodules form on compliant substrates through the active secretion of bone matrix via osteogenic AVICs [5,13,25]. These in vitro systems have helped identify many important mediators of dystrophic and osteogenic calcification; however, there exists ambiguity regarding what distinguishes each type of CNs [26]. The physicochemical composition and biophysical properties of the two nodule types remain largely undefined and their relationship to in vivo valvular calcification is unclear [13]. Clarifying the properties of CNs is necessary to more clearly delineate the two nodule types and provide a correlation between mechanistic changes and biophysical properties. Few studies have been conducted to describe the physicochemical properties of CNs. Cloyd et al. examined CNs formed using calcifying media supplemented with TGF-β1 with techniques such as SEM, transmission electron microscopy (TEM), and Raman spectroscopy [26]. Strikingly, these CNs were not mineralized but rather were rich in collagen content indicative of myofibroblast remodeling. This is in contrast to a study demonstrating, via TEM, that CNs formed in calcifying media without TGF-β1 contained a mineralized core [27]. Additionally, a seminal study investigating CN formation using infrared spectroscopy revealed that the CNs formed with TGF-β1 treatment exhibited a spectrum corresponding to the presence of hydroxyapatite in the nodule center [16]. Taken together, these studies suggest that all CNs are not equal and their properties are highly dependent on their culture conditions. Furthermore, these contrasting findings highlight the uncertainty that exists regarding CN properties and necessitate additional studies evaluating the physicochemical and biophysical properties of dystrophic and osteogenic CNs. In this study, we utilize SEM coupled with X-ray EDS (SEM-EDS) and AFM to define CN characteristics through two published in vitro systems of dystrophic and osteogenic calcification [13,19]. We found that both nodule types contained Ca and P contents; however, the regions where calcification forms were dramatically different. Dystrophic nodules had little to no surface calcification, whereas osteogenic nodules had an abundance of calcified spheres on the surface similar to what has been observed in vivo [28]. Also, both nodules had regions that did not contain calcification and exhibited modulus readings similar to that of cells. These data reveal a heterogeneous makeup of both nodules that have not been previously described. Furthermore, characteristics specific to dystrophic and osteogenic types were identified. Collectively, these findings ascribe unique characteristics to CN types and provide evidence connecting in vitro and in vivo valvular calcification.