Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease.

Statement of significance

We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease.

中文翻译：

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羟基磷灰石的结晶度驱动主动脉瓣膜中的肌纤维母细胞活化和钙化。

钙化主动脉瓣膜病（CAVD）是一种不可避免的变性病理，其特征是在瓣膜小叶上形成进行性钙化病变。晚期病变环境中瓣膜细胞的相互作用尚不十分清楚，但高度相关，因为临床上可检测到的CAVD表现出由非化学计量的羟基磷灰石（HA）组成的钙化。在这项研究中，傅里叶变换红外光谱成像用于空间分析矿物质特性与疾病进展的关系。结晶度（大小和完美度）随着瓣膜钙化的增加而增加。为了研究CAVD中结晶度与细胞行为之间的关系，将瓣膜细胞接种到含有合成HA颗粒的3D富含矿物质的胶原蛋白凝胶中，该凝胶具有不同的结晶度。较低的结晶度HA驱动瓣膜间质和内皮细胞中的肌纤维母细胞活化，以及间质细胞中的成骨细胞分化。此外，钙在凝胶中的积累取决于结晶度，而凋亡不足以解释HA驱动的细胞活性的差异。在较少结晶的HA颗粒的存在下，内皮细胞对间质细胞活化和钙积累的保护性质被完全抑制。阐明钙化后的瓣膜细胞行为对于更好地预测和治疗临床发病机制至关重要，含矿物质的水凝胶模型提供了独特的3D平台来评估瓣膜细胞对瓣膜疾病后期的反应。以及间质细胞中的成骨细胞分化。此外，钙在凝胶中的积累取决于结晶度，而凋亡不足以解释HA驱动的细胞活性的差异。在较少结晶的HA颗粒的存在下，内皮细胞对间质细胞活化和钙积累的保护性质被完全抑制。阐明钙化后的瓣膜细胞行为对于更好地预测和治疗临床发病机制至关重要，含矿物质的水凝胶模型提供了独特的3D平台来评估瓣膜细胞对瓣膜疾病后期的反应。以及间质细胞中的成骨细胞分化。此外，凝胶中钙的积累取决于结晶度，而凋亡不足以解释HA驱动的细胞活性的差异。在较少结晶的HA颗粒的存在下，内皮细胞对间质细胞活化和钙积累的保护性质被完全抑制。阐明钙化后的瓣膜细胞行为对于更好地预测和治疗临床发病机制至关重要，含矿物质的水凝胶模型提供了独特的3D平台来评估瓣膜细胞对瓣膜疾病后期的反应。细胞凋亡不足以解释HA驱动的细胞活性的差异。在较少结晶的HA颗粒的存在下，内皮细胞对间质细胞活化和钙积累的保护性质被完全抑制。阐明钙化后的瓣膜细胞行为对于更好地预测和治疗临床发病机制至关重要，含矿物质的水凝胶模型提供了独特的3D平台来评估瓣膜细胞对瓣膜疾病后期的反应。细胞凋亡不足以解释HA驱动的细胞活性的差异。在较少结晶的HA颗粒的存在下，内皮细胞对间质细胞活化和钙积累的保护性质被完全抑制。阐明钙化后的瓣膜细胞行为对于更好地预测和治疗临床发病机制至关重要，含矿物质的水凝胶模型提供了独特的3D平台来评估瓣膜细胞对瓣膜疾病后期的反应。

重要声明

我们在体外实施3D嵌入式羟基磷灰石（HA）纳米粒子的平台，以研究瓣膜间质细胞，瓣膜内皮细胞与富含矿物质的细胞外环境之间的相互作用。HA纳米颗粒的合成是基于对患病的人主动脉瓣钙化区域的矿物质特性的分析。我们的发现表明，HA的结晶度会驱动间质和内皮细胞的活化和分化。我们还表明，矿化的环境会阻止内皮细胞免受间质细胞钙化的影响。我们包含HA的水凝胶模型提供了一个独特的3D平台，用于评估瓣膜细胞对矿化ECM的反应。这项研究还为捕获钙化瓣膜中矿物质特性的多样性奠定了基础，并将这些特性与疾病的发展联系起来。