title: The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices.
abstract: The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium.
  
  
  OBJECTIVE
  This study evaluated the effect of different storage media on changes in matrix stiffness, loss of dry weight or solubilization of collagen from demineralized dentin beams incubated in vitro for up to 60 days.
  
  
  METHODS
  Dentin beams (1mm×2mm×6mm) were completely demineralized in 10% phosphoric acid. After baseline measurements of dry mass and elastic modulus (E) (3-point bending, 15% strain) the beams were divided into 5 groups (n=11/group) and incubated at 37°C in either media containing both zinc and calcium designated as complete medium (CM), calcium-free medium, zinc-free medium, a doubled-zinc medium or water. Beams were retested at 3, 7, 14, 30, and 60 days of incubation. The incubation media was hydrolyzed with HCl for the quantitation of hydroxyproline (HOP) as an index of solubilization of collagen by MMPs. Data were analyzed using repeated measures of ANOVA.
  
  
  RESULTS
  Both the storage medium and the storage time showed significant effects on E, mass loss and HOP release (p<0.05). The incubation in CM resulted in relatively rapid and significant (p<0.05) decreases in stiffness, and increasing amounts of mass loss. The HOP content of the experimental media also increased with incubation time but was significantly lower (p<0.05) than in the control CM medium, the recommended storage medium.
  
  
  CONCLUSIONS
  The storage solutions used to age resin-dentin bonds should be buffered solutions that contain both calcium and zinc. The common use of water as an aging medium may underestimate the hydrolytic activity of endogenous dentin MMPs.
authors.affiliation: Department of Prosthetic Dentistry, Institute of Dentistry, University of Turku, Finland. arztez@utu.fi
authors.forename: Arzu
authors.initials: A
authors.lastname: Tezvergil-Mutluay
authors.forename: Kelli A
authors.initials: KA
authors.lastname: Agee
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hoshika
authors.forename: Marcela
authors.initials: M
authors.lastname: Carrilho
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Breschi
authors.forename: Leo
authors.initials: L
authors.lastname: Tjäderhane
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nishitani
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Carvalho
authors.forename: Stephen
authors.initials: S
authors.lastname: Looney
authors.forename: Franklin R
authors.initials: FR
authors.lastname: Tay
authors.forename: David H
authors.initials: DH
authors.lastname: Pashley
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D003094:Collagen / Q000187:drug effects / Q000378:metabolism*; D003470:Culture Media; D003648:Decalcification Technique; D003804:Dentin / Q000187:drug effects / Q000378:metabolism*; D003808:Dentin Solubility / Q000187:drug effects; D003890:Desiccation; D055119:Elastic Modulus; D006801:Humans; D006868:Hydrolysis; D006909:Hydroxyproline / Q000032:analysis; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D010756:Phosphoric Acids; D018583:Pliability; D019252:Radiography, Dental, Digital; D013314:Stress, Mechanical; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D010756:Phosphoric Acids; D003094:Collagen; C030242:phosphoric acid; D020782:Matrix Metalloproteinases; D015032:Zinc; D006909:Hydroxyproline; D002118:Calcium