title: Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification.
abstract: 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 findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo.
  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 ¼. Particle nucleation 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.
authors.affiliation: Department of Toxicology, University of Zaragoza, Veterinary Faculty, Zaragoza, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Hortells
authors.affiliation: Department of Toxicology, University of Zaragoza, Veterinary Faculty, Zaragoza, Spain.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Sosa
authors.affiliation: Institute of Materials Science of Aragón, CSIC - Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Millán
authors.affiliation: Department of Toxicology, University of Zaragoza, Veterinary Faculty, Zaragoza, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: Sorribas
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D001639:Bicarbonates / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002130:Calcium Phosphates / Q000032:analysis / Q000378:metabolism*; D002245:Carbon Dioxide; D002417:Cattle; D018929:Cell Culture Techniques; D016923:Cell Death; D002478:Cells, Cultured; D011232:Chemical Precipitation; D003470:Culture Media / Q000494:pharmacology*; D005312:Fetal Blood; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D008297:Male; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012542:Scattering, Radiation; D061205:Vascular Calcification / Q000473:pathology*
chemical_list: D001639:Bicarbonates; D002130:Calcium Phosphates; D003470:Culture Media; D010710:Phosphates; C519480:amorphous calcium phosphate; D002245:Carbon Dioxide; D002118:Calcium