Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification

Hortells, Luis (Universidad de Zaragoza) ; Sosa, Cecilia (Universidad de Zaragoza) ; Millán, Ángel (Universidad de Zaragoza) ; Sorribas, Víctor (Universidad de Zaragoza)
Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification
Resumen: Background
Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide find- ings unrelated to VC. In this work we aimed to determine the critical experimental parame- ters that affect calcification in vitro and to determine the relevance to calcification in vivo.
Experimental Procedures and Results
Rat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca2+ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO2 vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to 1/4. Particle nucle- ation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO43--containing ACP.
The precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.

Idioma: Inglés
DOI: 10.1371/journal.pone.0141751
Año: 2015
Publicado en: PloS one 10, 11 (2015), e0141751 [22 p]
ISSN: 1932-6203

Factor impacto JCR: 3.057 (2015)
Categ. JCR: MULTIDISCIPLINARY SCIENCES rank: 11 / 62 = 0.177 (2015) - Q1 - T1
Factor impacto SCIMAGO: 1.427 - Agricultural and Biological Sciences (miscellaneous) (Q1) - Medicine (miscellaneous) (Q1) - Biochemistry, Genetics and Molecular Biology (miscellaneous) (Q1)

Financiación: info:eu-repo/grantAgreement/ES/MINECO/MAT2014-54975-R
Financiación: info:eu-repo/grantAgreement/ES/MINECO/SAF2012-33898
Tipo y forma: Article (Published version)
Área (Departamento): Area Toxicología (Dpto. Anat.Pat.Med.Leg.For.To.)
Área (Departamento): Área Física Materia Condensada (Dpto. Física Materia Condensa.)

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