000046951 001__ 46951
000046951 005__ 20180124100715.0
000046951 0247_ $$2doi$$a10.1371/journal.pone.0141751
000046951 0248_ $$2sideral$$a93350
000046951 037__ $$aART-2015-93350
000046951 041__ $$aeng
000046951 100__ $$0(orcid)0000-0003-1923-4918$$aHortells, Luis$$uUniversidad de Zaragoza
000046951 245__ $$aCritical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification
000046951 260__ $$c2015
000046951 5060_ $$aAccess copy available to the general public$$fUnrestricted
000046951 5203_ $$aBackground
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.
Conclusions
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.
000046951 536__ $$9info:eu-repo/grantAgreement/ES/MINECO/MAT2014-54975-R$$9info:eu-repo/grantAgreement/ES/MINECO/SAF2012-33898
000046951 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000046951 590__ $$a3.057$$b2015
000046951 591__ $$aMULTIDISCIPLINARY SCIENCES$$b11 / 60 = 0.183$$c2015$$dQ1$$eT1
000046951 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000046951 700__ $$0(orcid)0000-0003-2907-0427$$aSosa, Cecilia$$uUniversidad de Zaragoza
000046951 700__ $$0(orcid)0000-0003-0828-3212$$aMillán, Ángel$$uUniversidad de Zaragoza
000046951 700__ $$0(orcid)0000-0003-3457-323X$$aSorribas, Víctor$$uUniversidad de Zaragoza
000046951 7102_ $$11000$$2807$$aUniversidad de Zaragoza$$bDepartamento de Anatomía Patológica, Medicina Legal y Forense y Toxicología$$cToxicología
000046951 7102_ $$12003$$2395$$aUniversidad de Zaragoza$$bDepartamento de Física de la Materia Condensada$$cFísica de la Materia Condensada
000046951 773__ $$g10, 11 (2015), e0141751 [22 p]$$pPLoS One$$tPLoS One$$x1932-6203
000046951 8564_ $$s3989929$$uhttps://zaguan.unizar.es/record/46951/files/texto_completo.pdf$$yVersión publicada
000046951 8564_ $$s101611$$uhttps://zaguan.unizar.es/record/46951/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000046951 909CO $$ooai:zaguan.unizar.es:46951$$particulos$$pdriver
000046951 951__ $$a2018-01-24-09:59:32
000046951 980__ $$aARTICLE