No abstract

Objectives This study evaluated the role of endogenous dentin MMPs in auto-degradation of collagen fibrils within adhesive-bonded interfaces. The null hypotheses tested were that adhesive blends or chlorhexidine digluconate (CHX) application does not modify dentin MMPs activity and that CHX used as therapeutic primer does not improve the stability of adhesive interfaces over time. Methods Zymograms of protein extracts from human dentin powder incubated with Adper Scotchbond 1XT (SB1XT) on untreated or 0.2–2% CHX treated dentin were obtained to assay dentin MMPs activity. Microtensile bond strength and interfacial nanoleakage expression of SB1XT bonded interfaces (with or without CHX pre-treatment for 30s on the etched surface) were analyzed immediately and after 2 yr of storage in artificial saliva at 37°C. Results Zymograms showed that application of SB1XT to human dentin powder increases MMP-2 activity, while CHX pre-treatment inhibited all dentin gelatinolytic activity, irrespective from the tested concentration. CHX significantly lowered the loss of bond strength and nanoleakage seen in acid-etched resin-bonded dentin artificially aged for 2 yr. Significance The study demonstrates the active role of SB1XT in dentin MMP-2 activation and the efficacy of CHX inhibition of MMPs even if used at low concentration (0.2%).

Recent studies have reported collagen cross-linking after exposure to riboflavin followed by ultraviolet-A (UVA) exposure. This study is the first to investigate the effect of a riboflavin-containing primer on adhesive interface stability and dentinal matrix metalloproteinase activity. Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Adhesive was applied to control specimens without riboflavin/UVA. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy. To investigate dentinal matrix metalloproteinase activity, we performed correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond. Ultraviolet-activated riboflavin treatment increased the immediate bond strength to dentin at all aging intervals (p < 0.05 vs. control) and decreased interfacial nanoleakage in aged specimens (1 yr; p < 0.05). Zymograms revealed that riboflavin/UVA pre-treatment inhibited dentinal matrix metalloproteinase activity (especially MMP-9). In conclusion, dentinal collagen cross-linking induced by riboflavin/UVA increased immediate bond strength, stabilized the adhesive interface, and inhibited dentin matrix metalloproteinases, thereby increasing the durability of resin-dentin bonds.

Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin matrix and involved with degradation of the extracellular matrix components in hybrid layers and caries. Despite their identification through indirect evidences and biochemical assays, MMP-2 and -9 have not been localized within the human dentin extracellular organic matrix. Thus, this study aimed to assess the localization and distribution of MMP-2 and -9 in human dentin organic matrix by employing a correlative field emission in-lens-scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM) immunohistochemical approach. Dentin specimens were submitted either to a preembedding or to a postembedding immunolabeling technique using primary monoclonal antibodies anti-MMP-2 and anti-MMP-9 and exposed to a secondary antibody conjugated with gold nanoparticles. MMP-2 and -9 labelings were identified in the demineralized dentin matrix as highly electron-dense gold particles dispersed on the collagen fibrils. Correlative FEI-SEM/TEM observations confirmed that MMP-2 and MMP-9 are endogenous components of the human dentin organic matrix and revealed the three-dimensional relationship between these proteinases and the collagen fibrils, showing that both antibodies yielded a similar labeling pattern. In conclusion, the results of the study contribute to reveal distinct distribution pattern of gelatinases and support the hypothesis that these enzymes are intrinsic constituents of the dentin organic matrix after decalcification.

No abstract

SUMMARY Objective The function of endogenous MMP-3 and its distribution within the human dentin is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentin by means of biochemical and immunohistochemical assays. Methods Powdered dentin from extracted human teeth was prepared and 1) partially demineralized with 1% H3PO4 for 10 min or 2) untreated (control). The presence of MMP-3 was measured using a colorimetric assay system (QuantisirTM, Epigentek, USA). Additional cryo-fractured dentin fragments were processed for immunohistochemical identification of MMP-3 under FEI-SEM. Casein-zymography was used to investigate MMP-3 activity. Results MMP-3 detected level was 2.732 ng/µL in partially demineralized dentin powder, while it increased to 3.280 ng/µL in mineralized dentin. The FEI-SEM analysis revealed positive immunolabeling patterns for MMP-3, predominantly localized on the intertubular collagen fibrillar network showing MMP-3 directly or indirectly bound to the collagen fibrils. Casein-zymograms showed positive proteolytic activity for MMP-3 in demineralized dentin powder. Conclusion The results of the study clearly revealed the presence and distribution of MMP3 in human sound dentin. While the presence was verified, its role is still unclear. Future studies are needed to investigate the possible involvement of MMP-3 in physiological and pathological condition of the dentin-pulp complex.

Part I was an overview of the role and function of proteoglycans and glycoproteins in the pulpo–dentin complex; part II will focus on enzymes, serum proteins, and growth factors. This review will discuss current knowledge regarding matrix metalloproteinases (MMPs), cathepsins, serum proteins, and growth factors in dentin and the related dentin–pulp complex in an attempt to better understand their nature, role, and function in the dentin extracellular matrix (ECM) environment. Dentin formation in physiological and pathological conditions has been widely studied. However, the regulation and involvement of non‐collageneous enzymes, serum proteins, and growth factors are still not completely elucidated. MMPs, a family of 23 endopeptidases in humans, are collectively capable of degrading virtually all ECM components, and their specific tissue inhibitors (TIMPs: tissue inhibitors of matrix metalloproteinases) participate in organo‐ and morphogenesis, physiological tissue turnover, and pathological tissue destruction. Similarly, the lysosomal cysteine proteinases (cathepsins) are capable of degrading ECM proteins such as collagen, laminin, fibronectin, and proteoglycans. These enzymes are implicated in a variety of pathological conditions, especially in diseases involving tissue re‐modeling states. Dentin also contains serum‐derived proteins (such as albumin, immunoglobulins, and transferrin), and a variety of growth factors in the mineralized ECM are available for release during demineralization or other injury. A detailed description of the components of the above‐mentioned dentin non‐collageneous proteins will be summarized in this literature review.

The aim of this study was to compare the nanoleakage patterns of the resin-dentin interfaces of three dentin bonding systems at both TEM and field emission in lens SEM (FEI-SEM) levels. A standardized smear layer was created with 180-grit silicon carbide paper (SiC) on dentin disks obtained from 18 noncarious human third molars. Specimens were randomly divided into three groups and bonded with a two-step total etching adhesive (Single Bond, SB), a two-step, self-etching adhesive (Clearfil SE BOND, SEB), and a one-step, self-etching adhesive (XENO III, XEIII). Nanoleakage was evaluated by using an ammoniacal silver-nitrate solution. Specimens were processed for TEM and FEI-SEM observation. The TEM of SB revealed silver deposits in adhesive and hybrid layers (HL). High-magnification FEI-SEM micrographs clearly identified these deposits as spherical clusters mainly associated with nonembedded collagen fibrils. TEM and FEI-SEM examination of SEB revealed some clusters of silver deposits within porosities and small channels of the HL. Additional silver deposits were observed between the peritubular dentin walls and the resin tags. XEIII revealed very fine and diffuse silver grains throughout the entire HL. SEM visualization of nanoleakage at a high level of resolution has not been previously described. FEI-SEM technology supported the TEM visualization with three-dimensional morphological data of the relations between the HL constituents and nanoleakage. The results of the present study confirm the hypothesis that both total- and self-etch adhesives are not able to fully infiltrate the dentin substrate.