Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (∼20–28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.

Biochemical and immunohistochemical identification of MMP-7 in human dentin

Mazzoni, Annalisa;Maravić, Tatjana;Bavelloni, Alberto;Gobbi, Pietro;Breschi, Lorenzo
2018

Abstract

Objectives: Matrix metalloproteinases (MMPs) are dentinal endogenous enzymes claimed to have a vital role in dentin organic matrix breakdown. The aim of the study was to investigate presence, localization and distribution of MMP-7 in sound human dentin. Methods: Dentin was powdered, demineralized and dissolved in isoelectric focusing buffer. Resolved proteins were transferred to nitrocellulose membranes for western blotting (WB) analyses. For the zymographic analysis, aliquots of dentin protein were electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing fluorescently labeled gelatin. Further, the concentrations of dentinal MMPs were measured using Fluorescent Microsphere Immunoassay with a human MMP-MAP multiplex kit. Pre- and post-embedding immunolabeling technique was used to investigate the localization and distribution of MMP-7 in dentin. Dentin was cryo-fractured, the fragments partially decalcified and labeled with a primary monoclonal anti-MMP-7 and a secondary antibody conjugated with gold nanoparticles. MMP-7 labelings were identified in the demineralized dentin matrix as highly electron-dense dispersed gold particles. Results: WB and zymographic analysis of extracted dentin proteins showed presence of MMP-7 (∼20–28 KDa). Further, MMP-7 was found in the supernatants of the incubated dentin beams using Fluorescent Microsphere Immunoassay. FEI-SEM and TEM analyses established MMP-7 as an intrinsic constituent of the human dentin organic matrix. Conclusion: This study demonstrated that MMP-7 is an endogenous component of the human dentin fibrillar network. Clinical significance: It is pivotal to understand the underlying processes behind dentin matrix remodeling and degradation in order to develop the most optimal clinical protocols and ensure the longevity of dental restorations.
Mazzoni, Annalisa; Maravić, Tatjana; Tezvergil-Mutluay, Arzu; Tjäderhane, Leo; Scaffa, Polliana Mendes Candia; Seseogullari-Dirihan, Roda; Bavelloni, Alberto; Gobbi, Pietro; Pashley, David H.; Tay, Franklin R.; Breschi, Lorenzo*
File in questo prodotto:
Eventuali allegati, non sono esposti

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/655895
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 6
  • Scopus 5
  • ???jsp.display-item.citation.isi??? 6
social impact