Investigation of Mineral Components of Sclerotic Human Aortic Valve Tissue, Explanted Bio-prostheses and Calcifying Valvular Interstitial Cell Cultures by IR Spectroscopy

Objective: Stenotic aortic valve (AV) disease is mostly accompanied by calcification of leaflet tissue and the mineral phase consists of hydroxyapatite (HAP) and precursors. The molecular/cellular processes that lead to mineralization of original AV tissue are research focus. Therefore valvular interstitial cells (VICs) are cultured in vitro to investigate this process and to test substances that are able to stop disease progression. Aim of the project is to compare the chemical composition of in vitro calcification precipitates of human VIC culture model with HAP in original AV tissue by spectroscopic IR analysis. In addition calcified regions of explanted biological prostheses that can manifest several months after implantation are implemented. Investigation of lipid accumulation in AV structures is envisioned.

Methods: Sclerotic AV specimen, VIC culture pellets and an explanted bio-prosthesis were cryosectioned on a CaF₂ object slide. Technical features of IR spectroscopy: FT-IR spectrometer Vertex 70; infrared microscope Hyperion 3000; MCT focal plane array detector (64 × 64); 15-fold Cassegrainian objective (numerical aperture of 0.4); 6 cm⁻¹ spectral resolution. Matlab data evaluation considered the fingerprint region (950 to 1800 cm⁻¹). Parallel sections were histologically stained by Movats Pentachrom, van Kossa and HE.

Results: Analyzing an induced calcified VIC culture sample in comparison to a sclerotic AV tissue section reveals comparable spectral features with strong amide I, II and III bands at 1654, 1544 cm⁻¹ and 1237 cm⁻¹ and increased carbonyl stretching mode of phospholipid esters at 1744 cm⁻¹, respectively. Other bands located at 1402 and 1457 cm⁻¹ are assigned to CHx groups of lipids. Further spectra are dominated by a strong signal of hydroxyapatite phosphate groups of between 1000 to 1150 cm⁻¹ with less intensity of bands resulting from specimen tissue. Staining of parallel sections via van Kossa technique confirm calcification areas in the VIC culture pellet equivalent to sclerotic AV tissue section results and also in a AV prosthesis explanted three months after implantation. The respective prosthesis IR spectrum seems to differ considerably from AV sample results.

Conclusion: In vitro VIC culture model exhibits comparable IR spectra like an original sclerotic AV tissue. Therefore the induction model can be used to monitor molecular calcification processes and test substances. Calcification in AV prosthesis differs considerably.