Abstract Objectives Contemporary adhesives lose their bond strength to dentin regardless of the bonding system used. This loss relates to the hydrolysis of collagen matrix of the hybrid layers. The ...preservation of the collagen matrix integrity is a key issue in the attempts to improve the dentin bonding durability. Methods Dentin contains collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, which are responsible for the hydrolytic degradation of collagen matrix in the bonded interface. Results The identities, roles and function of collagenolytic enzymes in mineralized dentin has been gathered only within last 15 years, but they have already been demonstrated to have an important role in dental hard tissue pathologies, including the degradation of the hybrid layer. Identifying responsible enzymes facilitates the development of new, more efficient methods to improve the stability of dentin–adhesive bond and durability of bond strength. Significance Understanding the nature and role of proteolytic degradation of dentin–adhesive interfaces has improved immensely and has practically grown to a scientific field of its own within only 10 years, holding excellent promise that stable resin–dentin bonds will be routinely available in a daily clinical setting already in a near future.
Abstract Objective Endogenous dentin collagenolytic enzymes, matrix metalloproteinases (MMPs) and cysteine cathepsins, are responsible for the time-dependent hydrolysis of collagen matrix of hybrid ...layers. As collagen matrix integrity is essential for the preservation of long-term dentin bond strength, inhibition of endogenous dentin proteases is necessary for durable resin-bonded restorations. Methods Several tentative approaches to prevent enzyme function have been proposed. Some of them have already demonstrated clinical efficacy, while others need to be researched further before clinical protocols can be proposed. This review will examine both the principles and outcomes of techniques to prevent collagen hydrolysis in dentin–resin interfaces. Results Chlorhexidine, a general inhibitor of MMPs and cysteine cathepsins, is the most tested method. In general, these experiments have shown that enzyme inhibition is a promising approach to improve hybrid layer preservation and bond strength durability. Other enzyme inhibitors, e.g. enzyme-inhibiting monomers, may be considered promising alternatives that would allow more simple clinical application than chlorhexidine. Cross-linking collagen and/or dentin matrix-bound enzymes could render hybrid layer organic matrices resistant to degradation. Alternatively, complete removal of water from the hybrid layer with ethanol wet bonding or biomimetic remineralization should eliminate hydrolysis of both collagen and resin components. Significance Understanding the function of the enzymes responsible for the hydrolysis of hybrid layer collagen has prompted several innovative approaches to retain hybrid layer integrity and strong dentin bonding. The ultimate goal, prevention of collagen matrix degradation with clinically applicable techniques and commercially available materials may be achievable in several ways.
Abstract Introduction Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading ...enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-β) on cathepsin expression was also analyzed. Methods Human odontoblasts and pulp tissue were cultured with and without TGF-β, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. Results Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-β. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. Conclusions These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging.
Abstract Objective Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are known to be produced by MMPs. CTX fragments are thought to come from cathepsin K ...activity. The purpose of this study was to determine if quaternary methacrylates (QAMs) can inhibit matrix MMPs and cathepsins. Methods Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24 h at pH 7.4 to identify which cathepsins release CTX at neutral pH. Beams were dipped in ATA, an antimicrobial QAM to determine if it can inhibit dentin matrix proteases. Other beams were dipped in another QAM (MDPB) to determine if it produced similar inhibition of dentin proteases. Results Only beams incubated with cathepsin K lost more dry mass than the controls and released CTX. Dentin beams dipped in ATA and incubated for 1 week at pH 7.4, showed a concentration-dependent reduction in weight-loss. There was no change in ICTP release from control values, meaning that ATA did not inhibit MMPs. Media concentrations of CTX fell significantly at 15 wt% ATA indicating that ATA inhibits capthesins. Beams dipped in increasing concentrations of MDPB lost progressively less mass, showing that MDPB is a protease-inhibitor. ICTP released from controls or beams exposed to low concentrations were the same, while 5 or 10% MDPB significantly lowered ICTP production. CTX levels were strongly inhibited by 2.5–10% MDPB, indicating that MDPB is a potent inhibitor of both MMPs and cathepsin K. Significance CTX seems to be released from dentin matrix only by cathepsin K. MMPs and cathepsin K and B may all contribute to matrix degradation.
Abstract Objective The objective was to examine the effect of a solvent dimethyl sulfoxide (DMSO) on resin–dentin bond durability, as well as potential functional mechanisms behind the effect. ...Methods Microtensile bond strength (μTBS) was evaluated in extracted human teeth in two separate experiments. Dentin specimens were acid-etched and assigned to pre-treatment with 0.5 mM (0.004%) DMSO as additional primer for 30 s and to controls with water pre-treatment. Two-step etch-and-rinse adhesive (Scotchbond 1XT, 3M ESPE) was applied and resin composite build-ups were created. Specimens were immediately tested for μTBS or stored in artificial saliva for 6 and 12 months prior to testing. Additional immediate and 6-month specimens were examined for interfacial nanoleakage analysis under SEM. Matrix metalloproteinase (MMP) inhibition by DMSO was examined with gelatin zymography. Demineralized dentin disks were incubated in 100% DMSO to observe the optical clearing effect. Results The use of 0.5 mM DMSO had no effect on immediate bond strength or nanoleakage. In controls, μTBS decreased significantly after storage, but increased significantly in DMSO-treated group. The control group had significantly lower μTBS than DMSO-group after 6 and 12 months. DMSO also eliminated the increase in nanoleakage seen in controls. 5% and higher DMSO concentrations significantly inhibited the gelatinases. DMSO induced optical clearing effect demonstrating collagen dissociation. Significance DMSO as a solvent may be useful in improving the preservation of long-term dentin–adhesive bond strength. The effect may relate to dentinal enzyme inhibition or improved wetting of collagen by adhesives. The collagen dissociation required much higher DMSO concentrations than the 0.5 mM DMSO used for bonding.
This study investigated the effect of application of non‐thermal atmospheric plasma (NTAP) on the topography and composition of the dentin surface, as well as the microtensile bond strength (μTBS) of ...a universal adhesive to NTAP‐treated dentin. Exposed flat dentin surfaces from human third molars were either treated with NTAP for 10 and 30 s or untreated (control). The dentin‐surface topography and chemical composition were characterized by atomic force microscopy (n = 3) and Raman confocal spectroscopy (n = 5), respectively. The μTBS (n = 8) of Scotchbond Universal to dentin was determined after storage for 24 h and 1 yr, either by direct water exposure or under simulated pulpal pressure. In‐situ zymography was used to evaluate the influence of NTAP on the dentin‐enzymatic activity. Non‐thermal atmospheric plasma produced no remarkable topographical or chemical alterations at the dentin surface; only the amount of phosphate decreased following 10 s of treatment with NTAP. After 1 yr of direct water exposure, the μTBS of NTAP‐treated specimens did not differ statistically significantly from that of untreated controls, whereas simulated pulpal pressure‐aging resulted in a significantly higher μTBS for NTAP‐treated dentin. The dentin‐enzymatic activity appeared to be treatment‐dependent, but the untreated controls showed more intense fluorescence within the hybrid layer. Scotchbond Universal maintained its μTBS strength after 1 yr of direct water exposure and simulated pulpal pressure, although remarkable statistical differences between treatments were observed depending on the aging condition.
Crotamine, one of the main toxic components of
Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. It is ...well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5
min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells.
Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca2+ homeostasis, and apoptosis. Altered mitochondrial morphology and function ...is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.
•Why Cathepsins co-exist with MMPs in mature dentin matrix is presently unknown.•Leading activity of one group of protease over other is dependent on pH.•Along with MMPs, cathepsin B can exert ...proteolysis of dentin matrix at neutral pH.
Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH.
Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates’ cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B.
Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity.
Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.
The aim of this work was to evaluate the role of human plasma prekallikrein assembly and processing in cells and to determine whether proteoglycans, along with high molecular weight kininogen ...(H-kininogen), influence this interaction.
We used the endothelial cell line ECV304 and the epithelial cell lines CHO-K1 (wild type) and CHO-745 (deficient in proteoglycans). Prekallikrein endocytosis was studied using confocal microscopy, and prekallikrein cleavage/activation was determined by immunoblotting using an antibody directed to the prekallikrein sequence C364TTKTSTR371 and an antibody directed to the entire H-kininogen molecule.
At 37°C, prekallikrein endocytosis was assessed in the absence and presence of exogenously applied H-kininogen and found to be 1,418.4±0.010 and 1,070.3±0.001 pixels/cell, respectively, for ECV304 and 1,319.1±0.003 and 631.3±0.001 pixels/cell, respectively, for CHO-K1. No prekallikrein internalization was observed in CHO-745 in either condition. Prekallikrein colocalized with LysoTracker in the absence and presence of exogenous H-kininogen at levels of 76.0% and 88.5%, respectively, for ECV304 and at levels of 40.7% and 57.0%, respectively, for CHO-K1. After assembly on the cell surface, a plasma kallikrein fragment of 53 kDa was predominant in the incubation buffer of all the cell lines studied, indicating specific proteolysis; plasma kallikrein fragments of 48-44 kDa and 34-32 kDa were also detected in the incubation buffer, indicating non-specific cleavage. Bradykinin free H-kininogen internalization was not detected in CHO-K1 or CHO-745 cells at 37°C.
The prekallikrein interaction with the cell surface is temperature-dependent and independent of exogenously applied H-kininogen, which results in prekallikrein endocytosis promoted by proteoglycans. Prekallikrein proteolysis/activation is influenced by H-kininogen/glycosaminoglycans assembly and controls plasma kallikrein activity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
