title: Bone demineralization with citric acid enhances adhesion and spreading of preosteoblasts.
abstract: BACKGROUND
  Previous studies have demonstrated that bone demineralization can improve consolidation in bone grafts. The biologic mechanisms underlying this phenomenon remain unclear.
  
  
  METHODS
  Twelve adult male guinea pigs were used in this experiment. Forty-five bone samples removed from the calvaria of nine animals were divided in groups (n = 9) according to the time of demineralization with citric acid (50%, pH 1): 15, 30, 90, and 180 seconds and non-demineralized samples (control). Preosteoblasts (MC3T3-E1) were cultured on the bone samples for 24, 48, and 72 hours (n = 3). Fifteen samples removed from the remaining three animals were analyzed by scanning electron microscopy/energy dispersive spectrometry (SEM/EDS) after demineralization (n = 3).
  
  
  RESULTS
  The number of preosteoblasts increased significantly with time in all groups. The bone surface area covered by these cells increased with time, except in the control group. Intragroup differences occurred between 24 and 72 hours (P < 0.05). Samples demineralized for 30 seconds showed greater area covered by preosteoblast cells than for the other times of demineralization in all periods of cell culture (P < 0.05) without a statistically significant difference compared with 15 seconds. SEM/EDS showed diminished content of calcium (Ca) after 15 seconds of demineralization, but the Ca content increased after 180 seconds of demineralization (P < 0.05). The phosphorus (P) amount increased significantly only after 30 seconds of demineralization (P < 0.5). The sulfur (S) content was increased in demineralized samples in relation to non-demineralized ones, reaching the highest level after 90 seconds, when the difference became significant in relation to all the other times of demineralization (P < 0.05). Magnesium (Mg) content did not differ significantly between demineralized and non-demineralized samples.
  
  
  CONCLUSIONS
  Bone surfaces demineralized for 30 seconds increased the spreading of preosteoblasts as well as the surface area covered by these cells. Bone demineralization deserves to be studied in periodontal and maxillofacial regenerative procedures.
authors.affiliation: Department of Prosthodontics, Division of Periodontics, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Maria Lúcia R
authors.initials: ML
authors.lastname: de Rezende
authors.forename: Pedro T G
authors.initials: PT
authors.lastname: Coesta
authors.forename: Rodrigo C
authors.initials: RC
authors.lastname: de Oliveira
authors.forename: Samira
authors.initials: S
authors.lastname: Salmeron
authors.forename: Adriana C P
authors.initials: AC
authors.lastname: Sant'Ana
authors.forename: Carla A
authors.initials: CA
authors.lastname: Damante
authors.forename: Sebastião L A
authors.initials: SL
authors.lastname: Greghi
authors.forename: Alberto
authors.initials: A
authors.lastname: Consolaro
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018775:Bone Demineralization Technique / Q000379:methods*; D001842:Bone and Bones / Q000737:chemistry / Q000187:drug effects*; D002118:Calcium / Q000032:analysis; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D019343:Citric Acid / Q000494:pharmacology*; D003593:Cytoplasm / Q000648:ultrastructure; D006168:Guinea Pigs; D008274:Magnesium / Q000032:analysis; D008297:Male; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010006:Osteoblasts / Q000502:physiology*; D010758:Phosphorus / Q000032:analysis; D013052:Spectrometry, X-Ray Emission; D013455:Sulfur / Q000032:analysis; D013997:Time Factors; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D010758:Phosphorus; D019343:Citric Acid; D013455:Sulfur; D008274:Magnesium; D002118:Calcium
keywords: Bone tissue; cell adhesion; cells; citric acid; cultured; osteoblasts; spectrometry; x-ray emission