title: Use of crosslinkers to inactivate dentin MMPs.
abstract: OBJECTIVE
  This study evaluated the endogenous matrix metalloproteinase (MMP) activity of demineralized dentin matrix following 1 or 5 min pretreatment by various collagen crosslinkers. Generic MMP activity assay, total protein analysis, in situ zymography, gelatin zymography and multiplex bead technology were used to evaluate matrix-bound MMP activity.
  
  
  METHODS
  Six different crosslinkers; glutaraldehyde, riboflavin/UVA, riboflavin-5-monophospate/UVA, sumac berry extract, grape seed extract, and curcumin were used. Demineralized dentin beams were pretreated with respective crosslinkers for 1 or 5 min. Demineralized dentin beams with no crosslinker pretreatment served as control. The reduction in the total activity of dentin matrices were measured using generic MMP activity assay. Dentin slabs were used for in situ zymography and evaluated by using hydrolysis of self-quenched fluorescein-conjugated gelatin under confocal microscopy. Dentin beam extracts were used for total protein assay and multiplex analysis and powder extracts were used for gelatin zymography.
  
  
  RESULTS
  MMP activity in crosslinker pretreated samples decreased significantly between 21% and 70%, whereas untreated control samples' activity increased up to 84%. Zymograms confirmed a decrease in the gelatinolytic activity and in the amount of extractable total protein content. Multiplex analysis of extracts of crosslinker-treated dentin showed a reduction in the MMP-8, MMP-2 and MMP-9 release.
  
  
  CONCLUSIONS
  The result of this work suggests that the effect of the crosslinkers is source-dependent. The use of crosslinkers for as little as 1min on demineralized dentin can inactivate the endogenous protease activity of dentin matrices.
authors.affiliation: Finnish Doctoral Program in Oral Sciences (FINDOS) University of Turku, Institute of Dentistry, Turku, Finland.
authors.forename: R
authors.initials: R
authors.lastname: Seseogullari-Dirihan
authors.affiliation: Universidade Federal Do Ceara, Fortaleza, Brazil.
authors.forename: F
authors.initials: F
authors.lastname: Apollonio
authors.affiliation: Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna and IGM-CNR, Unit of Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Mazzoni
authors.affiliation: Department of Oral and Maxillofacial Diseases, University of Helsinki, and Helsinki University Hospital, Helsinki, Finland; Research Unit of Oral Health Sciences, and Medical Research Center Oulu (MRC Oulu), Oulu University Hospital and University of Oulu, Oulu, Finland.
authors.forename: L
authors.initials: L
authors.lastname: Tjaderhane
authors.affiliation: School of Dentistry, Georgia Regents University, Augusta, GA, USA.
authors.forename: D
authors.initials: D
authors.lastname: Pashley
authors.affiliation: Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna and IGM-CNR, Unit of Bologna, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Breschi
authors.affiliation: Department of Restorative Dentistry and Cariology, Adhesive Dentistry Research Group, Institute of Dentistry, University of Turku, Turku, Finland; Turku University Hospital, TYKS, University of Turku, Turku, Finland. Electronic address: arztez@utu.fi.
authors.forename: A
authors.initials: A
authors.lastname: Tezvergil-Mutluay
mesh_terms: D003124:Colorimetry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003474:Curcumin / Q000494:pharmacology; D003804:Dentin / Q000201:enzymology*; D057075:Enzyme Assays; D005976:Glutaral / Q000494:pharmacology; D056604:Grape Seed Extract / Q000494:pharmacology; D006801:Humans; D007118:Immunoassay; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D018613:Microscopy, Confocal; D010936:Plant Extracts / Q000494:pharmacology; D029045:Rhus; D012256:Riboflavin / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D056604:Grape Seed Extract; D010936:Plant Extracts; D020782:Matrix Metalloproteinases; D003474:Curcumin; D005976:Glutaral; D012256:Riboflavin
keywords: Collagen crosslinker; Cycteine cathepsins; Dentin; Gluteraldehyde; Matrix metalloproteinase