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Early Osteogenic Mineral Characteristics In Human Valvular Interstitial Cell Culture - Results Of IR-spectroscopy
Alice Mieting1, Gabriel Reimann1, Anett Jannasch1, Katrin Plötze1, Sems Malte Tugtekin1, Klaus Matschke1, Gerald Steiner2, Claudia Dittfeld1.
1Dresden University of Technology, Faculty of Medicine Carl Gustav Carus, Department of Cardiac Surgery, Herzzentrum Dresden, Dresden, Germany, 2Dresden University of Technology, Faculty of Medicine Carl Gustav Carus, Clinical Sensoring and Monitoring, Dresden, Germany.

OBJECTIVE:
The main pathological feature of Aortic valve (AV) stenosis is calcification. Molecular pathways leading to mineralization are research focus. The limitations of animal models point out the need for in vitro setups investigating human valvular interstitial cells (hVIC). Project aim is the detailed characterization of hVIC culture mineralization in comparison to AV calcification by IR-spectroscopy and therewith the validation of the in vitro setup for basic research.
METHODS:
hVICs were ADGMedium induced in vitro. Three VIC culture pellets and sclerotic AV tissue samples were cryosectioned on CaF2 slides. IR-spectroscopy: FT-IR spectrometer Vertex 70; infrared microscope Hyperion 3000; MCT focal plane array detector. The spectral region between 950 and 1800 cm-1 was evaluated by Matlab. Different vibration modes of hydroxyapatite (HAP) phosphate groups were identified by curve fitting and 2nd derivative spectroscopy. Parallel sections were stained by HE and von Kossa.
RESULTS:
Initial datasets of in vitro calcified hVIC samples reveal IR absorption bands mainly centered at 1020, 1061 and 1112 cm-1 which are assigned to carbonate-substituted HAP. A band at 1095 cm-1 results from reduced carbonate incorporation in comparison to carbonate-substituted HAP of hAV tissue and correlates with a lower carbonate/phosphate ratio in IR-spectra of hVIC compared to hAV. Other IR absorption bands located at 1112 and 1140 cm-1 resulting from acid phosphate substitution in apatite crystal and a lower intensity of HAP at 1060 cm-1 lead to the assumption that octacalciumphosphate-like apatitic mineral deposits in culture. IR-spectroscopic images reveal a heterogeneously distribution of the extracted molecular information across the samples reflecting the heterogeneity of hVIC preparations.
CONCLUSIONS:
Increased acid phosphate incorporation is described for freshly precipitated bone minerals whereas higher carbonate content in HAP characterizes mature bone mineral. Therefore initially IR-spectroscopy analysis of calcified VIC culture reveals early stages of biomineralisation comparable to common bone formation process.


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