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Protocol For Inducing Severe Calcification In Valves
Andres Rodriguez, Amanda Barreto, Sharan Ramaswamy.
Florida International University, Miami, FL, USA.

OBJECTIVE: Other than heart valve repair or replacement, therapeutic interventions are lacking for the management of heart valve disease. Part of the obstacle lies in vivo valve models not being able to recapitulate the human response. Thus, we investigated a suitable method to engineer severely calcified human valve tissues, with preliminary work focused on aortic valve porcine tissues.
METHODS: In valve disease, valve interstitial cells (VICs) are involved in the secretion of the calcified matrix. Aortic valve porcine tissues were harvested and placed into a static culture environment with pro-calcific media (DMEM, CaCl2, NaH2PO4, and Inorganic Pyrophosphatase “IP”) for seven days to chemically induce severe calcification. Next, the valves were embedded into a mold for sectioning and histological work was conducted using alizarin red staining to assess the degree of the valve calcification.
RESULTS: The results suggest that the calcification procedure induced severe calcification on the aortic valve porcine tissues which were proven by using alizarin red staining to mark signs of severe calcification. These results show similar calcific morphologies to severely calcified valve tissues containing human VICs found in a clinical study. (Bogdanova, M, et al. (2019)).

CONCLUSIONS: Creating a suitable method to induce severe calcification in an engineered valve tissue model system that mimics the calcified structure of native severely calcified heart valves may be useful in the assessment of current and emerging therapeutics for severe calcific valve disease. The results from the calcification process are promising as the calcified porcine tissues shared similar morphology to that of the calcified human tissues. Future experiments would involve repeating the same methodology on human VICs seeded onto a suitable bio-scaffold for creation of an engineered human valve tissue model system to aid in therapeutic discovery for calcific valve disease.


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