Biochemical and immunohistochemical identification of MMP-7 in human dentin

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.

Biochemical and immunohistochemical analysis of tissue inhibitor of metalloproteinases-1 in human sound dentin

“…In the recent years, several studies have identified different MMPs in human dentin, such as MMP-2, -3, -9 [22], MMP-7 [33], MMP-8 [34], MMP-20 [35], and cysteine proteinases [36][37][38]. At the same time, important findings have been made to search for molecules that can modulate the degradation of collagen fibrils in dentin [39][40][41] or inhibit the action of MMPs in their proteolytic activity such as quaternary ammonium methacrylates [42,43], chlorhexidine [44][45][46][47][48], and other molecules with specific targeting on MMPs [49].…”

Effect of a Copaiba Oil-Based Dental Biomodifier on the Inhibition of Metalloproteinase in Adhesive Restoration

“…ey are secreted in the form of zymogens [19]. MMP-2 and MMP-9 are the most prevalent in dentin, the first being in greater quantity [6][7][8].…”

“…Metalloproteinases are activated calcium-dependent and zinc enzymes and are naturally trapped in mineralized dentin during odontogenesis. However, they can be activated during previous acid conditioning or in acidic monomers present in the selfetching system [4][5][6][7][8].…”

Optical and Mechanical Properties of Highly Translucent Dental Zirconia

“…Fluorescence is the optical property, whereby a tooth absorbs ultraviolet (UV) light and then the activated electron returns to a lower energy level, making it emit blue light (440-490 nm) in the visible light spectrum [19,20]. Tooth dentin was found to be more fluorescent than enamel due to photosensitive organic components (fluorophores) [21,22]. In addition to the tooth compositions, its structural features, such as dentinal tubules, secondary dentin, inter-globular spaces, enamel lamellae, or Hunter-Schreger bands in tooth enamel, could increase the intensity of fluorescence [21].…”

“…Tooth dentin was found to be more fluorescent than enamel due to photosensitive organic components (fluorophores) [21,22]. In addition to the tooth compositions, its structural features, such as dentinal tubules, secondary dentin, inter-globular spaces, enamel lamellae, or Hunter-Schreger bands in tooth enamel, could increase the intensity of fluorescence [21]. It was also reported that the intensity of tooth fluorescence increased with age, while it was not related to tooth types [19].…”