NaF Inhibits Matrix-Bound Cathepsin-Mediated Dentin Matrix Degradation.

Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) degrade the collagen fibrils of demineralized dentin. Sodium fluoride (NaF) has previously been shown to inhibit recombinant MMP-2 and MMP-9. This study aimed to evaluate the efficacy of NaF on the inhibition of dentin-bound MMPs and CCs. Dentin beams were completely demineralized in 10% phosphoric acid. The baseline total MMP activity and dry masses were measured. Beams were assigned to test groups based on similar MMP activity and dry mass (n = 10/group), and incubated in artificial saliva (control) or artificial saliva with NaF containing 6-238 mM fluoride for 1, 7 and 21 days. The dry mass loss and MMP activities were reassessed at each time point. The proteolytic activity was screened by gelatin zymography. ICTP and CTX released to the incubation medium were analyzed as indices of MMP and cathepsin K activity, respectively. The beams were examined under scanning electron microscopy. All NaF doses reduced the dry mass loss after 21 days (p < 0.05). NaF inhibition of the total MMP activity ranged between 5 and 80%. In gelatin zymography, the bands of MMP-2 and MMP-9 became less prominent with increasing NaF levels. NaF did not decrease the released ICTP (p > 0.05). Less CTX release was detected with F ≥179 mM (p < 0.05). CaF2-like minerals were observed on the beams. High levels of NaF may slow the degradation of the dentin matrix due to the inhibition of cathepsin K. Fluoride does not seem effective in the direct inhibition of proteolysis by dentin matrix-bound MMPs.

Comparative study of demineralized collagen degradation determined by hydroxyproline assay and microscopic depth measurement.

INTRODUCTION: Quantification of collagen degradation is an important parameter to evaluate dentin caries progression or the efficacy of caries prevention aid. The aim of this study was to validate the simple light microscopic technique (LM) to evaluate collagen degradation by comparing with hydroxyproline assay technique (HPN). MATERIALS AND METHODS: Bovine root dentin blocks were embedded in acrylic resin, polished and covered with nail varnish except a 1.5 × 2.5mm window. The specimens were demineralized in acetate buffer (pH 4.3) for 3 days to create incipient lesions and were exposed to collagenase enzyme for 6, 9 and 16 h. The specimens were sectioned into thin sections (200-220 µm) to measure the degraded depth of collagen matrix by LM. The enzyme solutions were allocated to HPN assay using the simplified chloramines-T method. Correlation between LM and HPN was evaluated by Pearson correlation analysis. Anti-collagen degradation efficacy of 0.12% chlorhexidine (CHX) was evaluated by LM. RESULT: The depths of the degraded collagen and amount of hydroxyproline in 3 exposure periods were 27.8 ± 3.8 µm and 28.7 ± 4.2 µg for 6h, 48.1 ± 8.6 µm and 45.3 ± 6.1 µg for 9h, and 74.2 ± 9.7 µm and 71.3 ± 8.0 µg for 16 h, respectively. A significantly positive correlation (r=0.94, CI: 0.88-0.97, p<0.0001) was observed between LM and HPN and incubation time showed a linear correlation with amount of collagen degradation (R(2)=0.92). The CHX group (28.6 ± 3.3 µm) showed significantly lower collagen degradation than that of control group (53.1 ± 7.8 µm: p<0.01). CONCLUSION: The LM might be a reliable and simplified method to evaluate collagen degradation.

Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia.

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-ß-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.

The inhibitory effect of a polymerisable cationic monomer on functional matrix metalloproteinases.

OBJECTIVES: This study examined the use of methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) as a potential matrix metalloproteinases (MMPs) inhibitor on both soluble recombinant and dentine matrix-bound endogenous MMPs, meanwhile attempted to determine the effective anti-MMP group of quaternary ammonium methacrylates (QAMs). METHODS: The possible inhibitory effects of five DMAE-CB mass concentrations (0.1%, 0.5%, 1%, 3%, 5%) on soluble rhMMP-9 were measured using a colorimetic assay kit. Methyl methacrylate (MMA) and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METMAC) were also screened against rhMMP-9 to compare the inhibitory effect with DMAE-CB. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized dentine beams. Thirty beams were randomly divided into three groups (N=10) and respectively placed into 500µL of calcium- and zinc-containing media (CM; control), 0.2% chlorhexidine or 3% DMAE-CB in CM aged for 30 days. The changes in modulus of elasticity, loss of dry mass and solubilization of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. RESULTS: 0.5-5% mass concentrations of DMAE-CB were highly effective (P<0.05) in inhibiting rhMMP-9 varied between 76.56±6.44% and 97.06±3.24%, the inhibitory effect of MMA was much lower than that of METMAC and DMAE-CB at the same concentration (P<0.05). Dentine beams incubated in 3% DMAE-CB showed only a 26.34% decrease in the modulus of elasticity (75.74% decrease in control), a 1.72±1.56% loss of dry mass (29.70±9.12% loss in control), and significantly less solubilized hydroxyproline when compared with the control (P<0.05). CONCLUSIONS: DMAE-CB is effective in inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs. Quaternary ammonium group is the effective anti-MMP group of QAMs. CLINICAL SIGNIFICANCE: The incorporation of DMAE-CB into dental adhesives has the potential to enhance the durability of dentine bonding and thus increases the longevity of restorations.

In situ study of the gelatinase activity in demineralized dentin from rat molar teeth.

Matrix metalloproteinases (MMPs) in dentin are believed to participate in various physiological and pathological events in coronal dentin, but their exact source and location is not clear. The purpose of this study was to evaluate the activity of gelatinases in decalcified rat molars crowns by in situ zymography. Hemi-mandibles of five male Wistar rats were fixed in paraformaldehyde, decalcified in EDTA and glycerol solution and embedded in paraffin. Sections from the region of molar teeth were incubated with or without DQ gelatin in 50mM Tris-CaCl2 at 37°C for 2h and observed by means of confocal microscopy. Gelatinolytic activity was observed throughout the coronal dentin with varying intensities in different locations. High gelatinase activity was observed in the dentinal tubules, dentin-enamel junction (DEJ) and predentin, and it was weaker and less uniform in the intertubular dentin. This study shows that the location of gelatinase and relative activity can be detected by means of in situ zymography and confocal microcopy, and this methodology may provide a useful tool in studies on the role of gelatinases in tooth development, maturation and in pathological conditions.

Impact of protease inhibitors on dentin matrix degradation by collagenase.

This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 µM), chlorhexidine (CHX, 0.012%), FeSO(4) (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37°C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO(4) led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.

Distribution and relative activity of matrix metalloproteinase-2 in human coronal dentin.

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol x L(-1) EDTA/2 mol xL(-1) guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P = 0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD > ID > MD. Western blotting analysis detected -.66 and -72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a -66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD > ID > OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Effect of dentin etching and chlorhexidine application on metalloproteinase-mediated collagen degradation.

Dentin matrix metalloproteinases (MMPs) are involved in the degradation of collagen in resin-dentin interfaces. This study evaluated whether collagen degradation can be prevented by chlorhexidine digluconate (CHX) after different dentin demineralization procedures. The demineralization of human dentin was performed with phosphoric acid (PA), EDTA or acidic monomers (Clearfil SE Bond and Xeno V). Specimens were stored (for 24 h, or for 1 or 3 wk) in the presence or absence of CHX. In half of the groups, active MMP-2 was incorporated into the storage solution. At the end of each storage period, the C-terminal telopeptide (ICTP) concentration (which indicates the amount of collagen degradation) was measured in the storage solution. Collagen degradation was higher in PA- and EDTA-demineralized dentin. Chlorhexidine digluconate reduced collagen degradation in these groups only for 24 h. When dentin was demineralized with Clearfil SE Bond or Xeno V, collagen degradation was reduced by up to 30%, but the addition of exogenous MMP-2 significantly increased collagen degradation. In self-etchant-treated dentin, the inhibitory effect of CHX on MMPs lasted for up to 3 wk. Treating dentin with EDTA, PA or self-etching agents produces enough demineralization to permit cleavage of the exposed collagen. Monomer infiltration may exert protection on demineralized collagen, probably through immobilization of MMPs. The partial inhibitory action of CHX on MMP activity produced by self-etching adhesives was prolonged compared with the short-acting PA- or EDTA-treated dentin.

Influence of the digestive enzymes trypsin and pepsin in vitro on the progression of erosion in dentine.

OBJECTIVES: In patients with eating disorders, gastric and pancreatic enzymes could possibly reach the oral cavity during vomiting and could perhaps degrade the organic matrix of eroded dentine. This in vitro study sought to investigate whether pepsin, trypsin or the combination of both, have an influence on erosive mineral loss in dentine and whether they are able to degrade the organic matrix. METHODS: Sixty-four human dentine specimens were prepared and randomly divided into four groups. Specimens were cyclically de- and remineralised for six days. Demineralisation was performed with an HCl-solution (6x 5min daily, pH 1.6) in groups 1 and 3; in groups 2 and 4 the demineralisation solution additionally contained pepsin (750 microg/ml). After demineralisation, specimens of groups 3 and 4 were treated with a trypsin solution (6x 10min daily, 2000 BAEE/ml). After each day, mineral content (mum) was determined microradiographically, and the matrix degradation was determined by hydroxyproline analysis. RESULTS: After six days, treatment with pepsin (group 2) or trypsin (group 3) had no significant influence on mineral loss. The combined impact of pepsin and trypsin led to significantly higher mineral loss (group 4: 202.5+/-37.4) compared to all other groups (group 1: 139.1+/-29.5, p

Determination of matrix metalloproteinases in human radicular dentin.

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.

Effect of pepsin on erosive tissue loss and the efficacy of fluoridation measures in dentine in vitro.

OBJECTIVES: In dentine, erosive lesion progression and efficacy of fluoridation measures for symptomatic therapy of dental erosion are both dependent on the presence of the organic matrix. In patients with eating disorders in combination with chronic vomiting, the demineralized organic matrix can be degraded by gastric enzymes. The aim of this study was to investigate the effect of pepsin on erosion progression and the efficacy of fluoride in dentine. MATERIAL AND METHODS: Human dentine specimens were prepared and randomly divided into 4 groups of 20 specimens each. They were subjected to a cyclic de- and remineralization procedure for 9 days. For demineralization (6 x 2 min per day), an HCl solution (pH 1.6) was used in all groups. In two groups, pepsin (1.5 mg mL(-1)) was added to the demineralization solution. Fluoridation was performed in two groups 6 x 1 min per day with a mouth rinse (Olaflur/SnF(2); 250 ppm F(-)) after demineralization with both the HCl solution and the pepsin containing solution. Degradation of collagen was quantified by analyzing hydroxyproline and tissue loss was determined microradiographically. SEM images were taken in addition. RESULTS: In the pepsin group, 1.72 (0.26) microg mL(-1) (mean (SD)) hydroxyproline per day was detected, and in the pepsin-fluoride group 1.95 (0.50) microg mL(-1). Tissue loss after 9 days in the control group was similar to that in the pepsin group (122.2 (53.4) microm and 122.2 (38.0) microm, n.s., respectively). Fluoridation reduced tissue loss after demineralization (98.8 (30.2) microm) but not after pepsin treatment (125.2 (34.2) microm; p< or =0.05). CONCLUSION: Under the conditions used, pepsin had no influence on tissue loss, but altered the efficacy of fluoridation measures.

The role of matrix metalloproteinases in the oral environment.

This review focuses specifically on matrix metalloproteinases (MMPs) and their role in physiological and pathological extracellular matrix (ECM) remodeling and degradation processes in the oral environment. A group of enzymes capable of degrading almost all ECM proteins, MMPs contribute to both normal and pathological tissue remodeling. The expression of different MMPs may be upregulated in pathological conditions such as inflammation and tumor invasion. The balance between activated MMPs and tissue inhibitors of metalloproteinases (TIMPs) controls the extent of ECM remodeling. Prior to mineralization, MMPs may participate in the organization of enamel and dentin organic matrix, or they may regulate mineralization by controlling the proteoglycan turnover. There is evidence indicating that MMPs could be involved in the etiology of enamel fluorosis and amelogenesis imperfecta. They seem to play a part in dentinal caries progression, since they have a crucial role in dentin collagen breakdown in caries lesions. MMPs have been identified in pulpal and periapical inflammation and are strongly correlated with periodontal diseases, since they are the major players in collagen breakdown during periodontal tissue destruction. The use of MMP inhibitors could help the prevention and treatment of many MMP-related oral diseases.

The role of matrix metalloproteinases (MMPs) in human caries.

The objective of this review is to summarize our understanding of the role of host matrix metalloproteinases (MMPs) in the caries process and to discuss new therapeutic avenues. MMPs hydrolyze components of the extracellular matrix and play a central role in many biological and pathological processes. MMPs have been suggested to play an important role in the destruction of dentin organic matrix following demineralization by bacterial acids and, therefore, in the control or progression of carious decay. Host-derived MMPs can originate both from saliva and from dentin. They may be activated by an acidic pH brought about by lactate release from cariogenic bacteria. Once activated, they are able to digest demineralized dentin matrix after pH neutralization by salivary buffers. Furthermore, the degradation of SIBLINGs (Small Integrin-binding Ligand N-linked Glycoproteins) by the caries process may potentially enhance the release of MMPs and their activation. This review also explores the different available MMP inhibitors, natural or synthetic, and suggests that MMP inhibition by several inhibitors, particularly by natural substances, could provide a potential therapeutic pathway to limit caries progression in dentin.

Effects of collagenase on root demineralization.

The role of proteolytic enzymes in the root caries process remains unclear. The aim of this study was to investigate collagenase activity during tooth root demineralization and remineralization in an in vitro demineralization/remineralization pH-cycling model, Human tooth roots were subjected to pH cycling (alternating demineralization and remineralization) in one of two different time cycles for five days. Collagenase at 90, 180, or 360 micrograms per root was placed into either the demineralizing solution or the remineralizing solution in the pH-cycling system. The effects of additional exposure to collagenase before or after pH cycling were also studied. After the exposure, thin sections of the roots were examined histologically by polarized light microscopy. Changes of calcium and phosphate in the solutions were analyzed chemically. Surface erosion occurred only in the groups where collagenase was contained in the remineralizing solution and in which the root samples were exposed to severe demineralization. However, no differences among the control and experimental groups were found in calcium and phosphate changes in the pH-cycling solutions. These findings suggest that collagenase works during the remineralizing phase and predominantly attacks the organic matrix of the root after demineralization. Additional exposure to collagenase before or after pH cycling did not increase surface erosion except for exposure to collagenase in the absence of phosphate following pH cycling.