Objectives-While screening the activity of potential inhibitors of matrix metalloproteinases (MMP... more Objectives-While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols.
The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of col... more The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium.
ABSTRACT Matrix metalloproteinases (MMPs) are zinc dependent enzymes that are trapped within the ... more ABSTRACT Matrix metalloproteinases (MMPs) are zinc dependent enzymes that are trapped within the mineralized dentin matrix. Objective: The aim of this study was to evaluate the effect of phosphoric acid (PA) concentration and demineralization duration on the release of matrix-bound MMPs from dentin. Method: Ninety extracted third molars were used to prepare dentin beams. After baseline measurement of the dry mass, beams were divided into 9 groups (n=10/group) and demineralized in 10% or 37% phosphoric acid for 6, 12, 18, or 24 hrs. Dentin beams with no treatment served as control. After demineralization and assessment of dry weight, the beams were further incubated in 1mL calcium and zinc containing artificial saliva at 37 °C for one week and both the buffered demineralization media and the incubation media were quantitatively evaluated by fluorescent microsphere immunoassay with Fluorokine 1 MAP Multipleks kit (R&D Systems, USA) for the MMP content. Data were analyzed using ANOVA and Tukey’s test. Result: Phosphoric acid concentration did not have significant effect on the dry mass loss or the release of matrix-bound MMPs (p>0.05). Demineralization duration had significant effect (p<0.05) on the release of MMPs. Demineralization for 18 or 24 hrs resulted in significantly more MMP release compared to 6 or 12 hrs. MMP-9 release at 18-24 hrs ranged between 22.5 to 30 pg/mg dentin followed by MMP-8 (18 pg/mg dentin) and MMP-2 (17 pg/mg dentin), MMP-7 (14 pg/mgdentin, and MMP-3 (10 pg/mg dentin). MMP-9 release was significantly higher also at one week storage media compared to others (p<0.05). Conclusion: This study demonstrated that MMP-9 could be more easily extracted from dentin compared to other MMPs. The use of fluorescent microsphere immunoassay provides possibility to quantitate the release of matrix-bound MMPs.
Objective: Matrix metalloproteinases (MMPs) and cathepsins were both identified in human dentin a... more Objective: Matrix metalloproteinases (MMPs) and cathepsins were both identified in human dentin and in part responsible for the collagen degradation in hybrid layers created by dentin adhesives. The aim of this study was to evaluate the effect of phosphoric acid (PA) concentration and demineralization duration on the protease activity in dentin. Method: Ninety extracted third molars were used to prepare dentin beams. After baseline measurement of the dry mass, beams were divided into 9 groups (n=10/group) and demineralized in 10% or 37% PA for 6, 12, 18, or 24 hrs. The beams that were kept mineralized served as control. After demineralization and assessment of dry weight, the beams were incubated in 1mL calcium and zinc containing artificial saliva at 37 °C for 1, 3 and 7 days. After each incubation period aliquots of the incubation media were analyzed for pyridinoline-crosslink degradation fragment of the C-terminal telopeptide of type I collagen (ICTP) for MMP-mediated degradation...
j o u r n a l o f d e n t i s t r y 3 9 ( 2 0 1 1 ) 4 7 0 -4 7 7 Biochemistry Immunohistochemistr... more j o u r n a l o f d e n t i s t r y 3 9 ( 2 0 1 1 ) 4 7 0 -4 7 7 Biochemistry Immunohistochemistry a b s t r a c t Objective: Degradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive.
Dental materials : official publication of the Academy of Dental Materials, 2014
The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde an... more The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 s and to evaluate its effect on dentin matrix stiffness and dry mass weight. Dentin beams of 2 mm×1 mm×6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 s and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 s, rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 s. Additional beams of 1 mm×1 mm×6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity (E) after 60 s of Gluma treatment followed by incubation in simulated body fluid buffer for 0, 1 or 4 weeks. E was measur...
It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be... more It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be important in tooth development (1) and dentinal caries. Acids are also known to activate MMPs (2-5). Some of these MMPs (MMP-1, -8, -13) attack collagen, while others (MMP-2 and -9) attack gelatine. Dentin is known to contain MMP-2, MMP-20 and possibly MMP-8 (6, 7). Using fluorescein-labeled collagen, partially mineralized human dentin powder has been shown to have a low, but significant, collagenase activity, that seems to be responsible for the in vitro degradation of demineralized dentin over a 270-d time period (8) and may be responsible for the degradation of hybrid layers in vivo (9). However, the addition of four protease inhibitors to the incubation medium completely inhibited in vitro degradation of the dentin matrix over 270 d and inhibited the collagenolytic activity of mineralized dentin powder by 73% (8). In that same study, treatment of mineralized dentin powder with 37 weight percentage (wt%) phosphoric acid gel for 15 s, reduced the collagenolytic activity by 65% (8). It was thought that the low pH (10) of 37 wt% phosphoric acid gel (pH ¼ )0.17), while demineralizing the dentin powder and exposing more MMP activity, also denatured the enzymes. The residual collagenolytic activity was probably associated with the central portion of the powder that had not been demineralized in the 15-s etch.
Objectives-To better comprehend the role of CHX in the preservation of resin-dentin bonds, this s... more Objectives-To better comprehend the role of CHX in the preservation of resin-dentin bonds, this study investigated the substantivity of CHX to human dentin.
It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be... more It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be important in tooth development (1) and dentinal caries. Acids are also known to activate MMPs (2-5). Some of these MMPs (MMP-1, -8, -13) attack collagen, while others (MMP-2 and -9) attack gelatine. Dentin is known to contain MMP-2, MMP-20 and possibly MMP-8 (6, 7). Using fluorescein-labeled collagen, partially mineralized human dentin powder has been shown to have a low, but significant, collagenase activity, that seems to be responsible for the in vitro degradation of demineralized dentin over a 270-d time period (8) and may be responsible for the degradation of hybrid layers in vivo (9). However, the addition of four protease inhibitors to the incubation medium completely inhibited in vitro degradation of the dentin matrix over 270 d and inhibited the collagenolytic activity of mineralized dentin powder by 73% (8). In that same study, treatment of mineralized dentin powder with 37 weight percentage (wt%) phosphoric acid gel for 15 s, reduced the collagenolytic activity by 65% (8). It was thought that the low pH (10) of 37 wt% phosphoric acid gel (pH ¼ )0.17), while demineralizing the dentin powder and exposing more MMP activity, also denatured the enzymes. The residual collagenolytic activity was probably associated with the central portion of the powder that had not been demineralized in the 15-s etch.
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.co... more A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578 a v a i l a b l e a t w w w . s c i e n c e... more d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578
Dentine bonding agent Biochemical assay Immunohistochemistry a b s t r a c t Objective: The funct... more Dentine bonding agent Biochemical assay Immunohistochemistry a b s t r a c t Objective: The function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays.
Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in colla... more 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. (J Endod 2009;35:686-689)
Objectives-The purposes of this work were to quantitate the affinity and binding capacity of chlo... more Objectives-The purposes of this work were to quantitate the affinity and binding capacity of chlorhexidine (CHX) digluconate to mineralized vs. demineralized dentin powder, and to determine how much debinding would result from rinsing with water, ethanol, hydroxyethylmethacrylate (HEMA) or 0.5 M NaCl in water.
Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of h... more Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of host-derived matrix metalloproteinases. As phosphoric acid-etching inactivates these endogenous enzymes, it is puzzling how hybrid layers created by simplified etch-and-rinse adhesives can degrade in vivo. This study tested the null hypothesis that there are no differences in the relative proteolytic activities of mineralised dentine, acid-etched dentine, and etch-and-rinse adhesivetreated acid-etched dentine. Powdered dentine prepared from extracted human teeth was treated with 17% EDTA, 10% phosphoric acid, or with five simplified etch-and-rinse adhesives that were applied to 10% phosphoric acid-etched dentine. The gelatinolytic activity of the dentine powder was assayed using fluorescein-labelled gelatine. TEM examination of the air-dried, treated dentine powder was performed to confirm the presence of remnant mineralised dentine after acid-etching. 17% EDTA significantly reduced the relative proteolytic activity (73.2%) of the untreated mineralised dentine powder (control), while 10% phosphoric acid-etched dentine exhibited the highest reduction (98.1%). Treating the acid-etched dentine powder with any of the five simplified etch-and-rinse adhesives resulted in the reactivation of the proteolytic activity, with a significant negative linear correlation (Po0:05) between the increases in fluorescence and the corresponding pH values of the adhesives. It is concluded that simplified etch-and-rinse adhesives can reactivate endogenous enzymatic activities in dentine that are previously inactivated by phosphoric acid-etching. The amount of enzyme reactivated may even exceed the original quantity present in untreated mineralised dentine. This provides an explanation for the degradation of hybrid layers after acid-etched dentine matrices are infiltrated with these adhesives. r
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2008
Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding p... more Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding procedures and are thought to contribute to the progressive degradation of resin-dentin bonds over time. The purpose of this study was to evaluate the changes in mechanical, biochemical and structural properties of demineralized dentin treated with or without chlorhexidine (CHX), a known MMP-inhibitor. After demineralizing dentin beams in EDTA or phosphoric acid (PA), the baseline modulus of elasticity (E) of each beam was measured by 3point flexure. Specimens were pretreated with water (control) or with 2% CHX (experimental) and then incubated in artificial saliva (AS) at 37°C for 4 weeks. The E of each specimen was remeasured weekly and, the media was analyzed for solubilized dentin collagen at first and fourth week of incubation. Some specimens were processed for electron microscopy (TEM) immediately after demineralization and after 4 weeks of incubation. In EDTA and PA-demineralized specimens, the E of the control specimens fell (p<0.05) after incubation in AS, while there were no changes in E in the CHX-pretreated specimens over time. More collagen was solubilized from PA-demineralized controls (p<0.05) than from EDTA-demineralized matrices after 1 or 4 weeks. Less collagen (p<0.05) was solubilized from CHX-pretreated specimens demineralized in EDTA compared to PA. TEM examination of control beams revealed that prolonged demineralization of dentin in 10% PA (12 h) did not denature the collagen fibrils.
Journal of Biomedical Materials Research Part A, 2009
Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin mat... more 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.
Dental materials : official publication of the Academy of Dental Materials, 2015
Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are kn... more 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. Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24h 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. 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 ...
Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-in... more Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-infiltrated dentin. This study tested the hypothesis that interfacial degradation of resin-dentin bonds may be prevented or delayed by the application of chlorhexidine (CHX), a matrix metalloproteinase inhibitor, to dentin after phosphoric acid-etching. Contralateral pairs of resin-bonded Class I restorations in non-carious third molars were kept under intra-oral function for 14 months. Preservation of resin-dentin bonds was assessed by microtensile bond strength tests and TEM examination. In vivo bond strength remained stable in the CHX-treated specimens, while bond strength decreased significantly in control teeth. Resin-infiltrated dentin in CHX-treated specimens exhibited normal structural integrity of the collagen network. Conversely, progressive disintegration of the fibrillar network was identified in control specimens. Auto-degradation of collagen matrices can occur in resin-infiltrated dentin, but may be prevented by the application of a synthetic protease inhibitor, such as chlorhexidine.
The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that... more The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2&amp;amp;amp;amp;amp;amp;amp;amp;#39; of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.
Objectives-While screening the activity of potential inhibitors of matrix metalloproteinases (MMP... more Objectives-While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols.
The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of col... more The progressive degradation of resin-dentin bonds is due, in part, to the slow degradation of collagen fibrils in the hybrid layer by endogenous matrix metalloproteinases (MMPs) of the dentin matrix. In in vitro durability studies, the storage medium composition might be important because the optimum activity of MMPs requires both zinc and calcium.
ABSTRACT Matrix metalloproteinases (MMPs) are zinc dependent enzymes that are trapped within the ... more ABSTRACT Matrix metalloproteinases (MMPs) are zinc dependent enzymes that are trapped within the mineralized dentin matrix. Objective: The aim of this study was to evaluate the effect of phosphoric acid (PA) concentration and demineralization duration on the release of matrix-bound MMPs from dentin. Method: Ninety extracted third molars were used to prepare dentin beams. After baseline measurement of the dry mass, beams were divided into 9 groups (n=10/group) and demineralized in 10% or 37% phosphoric acid for 6, 12, 18, or 24 hrs. Dentin beams with no treatment served as control. After demineralization and assessment of dry weight, the beams were further incubated in 1mL calcium and zinc containing artificial saliva at 37 °C for one week and both the buffered demineralization media and the incubation media were quantitatively evaluated by fluorescent microsphere immunoassay with Fluorokine 1 MAP Multipleks kit (R&amp;D Systems, USA) for the MMP content. Data were analyzed using ANOVA and Tukey’s test. Result: Phosphoric acid concentration did not have significant effect on the dry mass loss or the release of matrix-bound MMPs (p&gt;0.05). Demineralization duration had significant effect (p&lt;0.05) on the release of MMPs. Demineralization for 18 or 24 hrs resulted in significantly more MMP release compared to 6 or 12 hrs. MMP-9 release at 18-24 hrs ranged between 22.5 to 30 pg/mg dentin followed by MMP-8 (18 pg/mg dentin) and MMP-2 (17 pg/mg dentin), MMP-7 (14 pg/mgdentin, and MMP-3 (10 pg/mg dentin). MMP-9 release was significantly higher also at one week storage media compared to others (p&lt;0.05). Conclusion: This study demonstrated that MMP-9 could be more easily extracted from dentin compared to other MMPs. The use of fluorescent microsphere immunoassay provides possibility to quantitate the release of matrix-bound MMPs.
Objective: Matrix metalloproteinases (MMPs) and cathepsins were both identified in human dentin a... more Objective: Matrix metalloproteinases (MMPs) and cathepsins were both identified in human dentin and in part responsible for the collagen degradation in hybrid layers created by dentin adhesives. The aim of this study was to evaluate the effect of phosphoric acid (PA) concentration and demineralization duration on the protease activity in dentin. Method: Ninety extracted third molars were used to prepare dentin beams. After baseline measurement of the dry mass, beams were divided into 9 groups (n=10/group) and demineralized in 10% or 37% PA for 6, 12, 18, or 24 hrs. The beams that were kept mineralized served as control. After demineralization and assessment of dry weight, the beams were incubated in 1mL calcium and zinc containing artificial saliva at 37 °C for 1, 3 and 7 days. After each incubation period aliquots of the incubation media were analyzed for pyridinoline-crosslink degradation fragment of the C-terminal telopeptide of type I collagen (ICTP) for MMP-mediated degradation...
j o u r n a l o f d e n t i s t r y 3 9 ( 2 0 1 1 ) 4 7 0 -4 7 7 Biochemistry Immunohistochemistr... more j o u r n a l o f d e n t i s t r y 3 9 ( 2 0 1 1 ) 4 7 0 -4 7 7 Biochemistry Immunohistochemistry a b s t r a c t Objective: Degradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive.
Dental materials : official publication of the Academy of Dental Materials, 2014
The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde an... more The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 s and to evaluate its effect on dentin matrix stiffness and dry mass weight. Dentin beams of 2 mm×1 mm×6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 s and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 s, rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 s. Additional beams of 1 mm×1 mm×6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity (E) after 60 s of Gluma treatment followed by incubation in simulated body fluid buffer for 0, 1 or 4 weeks. E was measur...
It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be... more It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be important in tooth development (1) and dentinal caries. Acids are also known to activate MMPs (2-5). Some of these MMPs (MMP-1, -8, -13) attack collagen, while others (MMP-2 and -9) attack gelatine. Dentin is known to contain MMP-2, MMP-20 and possibly MMP-8 (6, 7). Using fluorescein-labeled collagen, partially mineralized human dentin powder has been shown to have a low, but significant, collagenase activity, that seems to be responsible for the in vitro degradation of demineralized dentin over a 270-d time period (8) and may be responsible for the degradation of hybrid layers in vivo (9). However, the addition of four protease inhibitors to the incubation medium completely inhibited in vitro degradation of the dentin matrix over 270 d and inhibited the collagenolytic activity of mineralized dentin powder by 73% (8). In that same study, treatment of mineralized dentin powder with 37 weight percentage (wt%) phosphoric acid gel for 15 s, reduced the collagenolytic activity by 65% (8). It was thought that the low pH (10) of 37 wt% phosphoric acid gel (pH ¼ )0.17), while demineralizing the dentin powder and exposing more MMP activity, also denatured the enzymes. The residual collagenolytic activity was probably associated with the central portion of the powder that had not been demineralized in the 15-s etch.
Objectives-To better comprehend the role of CHX in the preservation of resin-dentin bonds, this s... more Objectives-To better comprehend the role of CHX in the preservation of resin-dentin bonds, this study investigated the substantivity of CHX to human dentin.
It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be... more It is well known that the dentin matrix contains matrix metalloproteinases (MMPs) that seem to be important in tooth development (1) and dentinal caries. Acids are also known to activate MMPs (2-5). Some of these MMPs (MMP-1, -8, -13) attack collagen, while others (MMP-2 and -9) attack gelatine. Dentin is known to contain MMP-2, MMP-20 and possibly MMP-8 (6, 7). Using fluorescein-labeled collagen, partially mineralized human dentin powder has been shown to have a low, but significant, collagenase activity, that seems to be responsible for the in vitro degradation of demineralized dentin over a 270-d time period (8) and may be responsible for the degradation of hybrid layers in vivo (9). However, the addition of four protease inhibitors to the incubation medium completely inhibited in vitro degradation of the dentin matrix over 270 d and inhibited the collagenolytic activity of mineralized dentin powder by 73% (8). In that same study, treatment of mineralized dentin powder with 37 weight percentage (wt%) phosphoric acid gel for 15 s, reduced the collagenolytic activity by 65% (8). It was thought that the low pH (10) of 37 wt% phosphoric acid gel (pH ¼ )0.17), while demineralizing the dentin powder and exposing more MMP activity, also denatured the enzymes. The residual collagenolytic activity was probably associated with the central portion of the powder that had not been demineralized in the 15-s etch.
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.co... more A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578 a v a i l a b l e a t w w w . s c i e n c e... more d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 571-578
Dentine bonding agent Biochemical assay Immunohistochemistry a b s t r a c t Objective: The funct... more Dentine bonding agent Biochemical assay Immunohistochemistry a b s t r a c t Objective: The function of endogenous MMP-3 and its distribution within the human dentine is unclear. Thus, the aim of the present study was to assay the presence and distribution of MMP-3 within human sound dentine by means of biochemical and immunohistochemical assays.
Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in colla... more 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. (J Endod 2009;35:686-689)
Objectives-The purposes of this work were to quantitate the affinity and binding capacity of chlo... more Objectives-The purposes of this work were to quantitate the affinity and binding capacity of chlorhexidine (CHX) digluconate to mineralized vs. demineralized dentin powder, and to determine how much debinding would result from rinsing with water, ethanol, hydroxyethylmethacrylate (HEMA) or 0.5 M NaCl in water.
Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of h... more Auto-degradation of collagen matrices occurs in resin-infiltrated dentine by the slow action of host-derived matrix metalloproteinases. As phosphoric acid-etching inactivates these endogenous enzymes, it is puzzling how hybrid layers created by simplified etch-and-rinse adhesives can degrade in vivo. This study tested the null hypothesis that there are no differences in the relative proteolytic activities of mineralised dentine, acid-etched dentine, and etch-and-rinse adhesivetreated acid-etched dentine. Powdered dentine prepared from extracted human teeth was treated with 17% EDTA, 10% phosphoric acid, or with five simplified etch-and-rinse adhesives that were applied to 10% phosphoric acid-etched dentine. The gelatinolytic activity of the dentine powder was assayed using fluorescein-labelled gelatine. TEM examination of the air-dried, treated dentine powder was performed to confirm the presence of remnant mineralised dentine after acid-etching. 17% EDTA significantly reduced the relative proteolytic activity (73.2%) of the untreated mineralised dentine powder (control), while 10% phosphoric acid-etched dentine exhibited the highest reduction (98.1%). Treating the acid-etched dentine powder with any of the five simplified etch-and-rinse adhesives resulted in the reactivation of the proteolytic activity, with a significant negative linear correlation (Po0:05) between the increases in fluorescence and the corresponding pH values of the adhesives. It is concluded that simplified etch-and-rinse adhesives can reactivate endogenous enzymatic activities in dentine that are previously inactivated by phosphoric acid-etching. The amount of enzyme reactivated may even exceed the original quantity present in untreated mineralised dentine. This provides an explanation for the degradation of hybrid layers after acid-etched dentine matrices are infiltrated with these adhesives. r
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2008
Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding p... more Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding procedures and are thought to contribute to the progressive degradation of resin-dentin bonds over time. The purpose of this study was to evaluate the changes in mechanical, biochemical and structural properties of demineralized dentin treated with or without chlorhexidine (CHX), a known MMP-inhibitor. After demineralizing dentin beams in EDTA or phosphoric acid (PA), the baseline modulus of elasticity (E) of each beam was measured by 3point flexure. Specimens were pretreated with water (control) or with 2% CHX (experimental) and then incubated in artificial saliva (AS) at 37°C for 4 weeks. The E of each specimen was remeasured weekly and, the media was analyzed for solubilized dentin collagen at first and fourth week of incubation. Some specimens were processed for electron microscopy (TEM) immediately after demineralization and after 4 weeks of incubation. In EDTA and PA-demineralized specimens, the E of the control specimens fell (p<0.05) after incubation in AS, while there were no changes in E in the CHX-pretreated specimens over time. More collagen was solubilized from PA-demineralized controls (p<0.05) than from EDTA-demineralized matrices after 1 or 4 weeks. Less collagen (p<0.05) was solubilized from CHX-pretreated specimens demineralized in EDTA compared to PA. TEM examination of control beams revealed that prolonged demineralization of dentin in 10% PA (12 h) did not denature the collagen fibrils.
Journal of Biomedical Materials Research Part A, 2009
Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin mat... more 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.
Dental materials : official publication of the Academy of Dental Materials, 2015
Dentin matrices release ICTP and CTX fragments during collagen degradation. ICTP fragments are kn... more 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. Dentin beams were demineralizated, and dried to constant weight. Beams were incubated with rh-cathepsin B, K, L or S for 24h 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. 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 ...
Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-in... more Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-infiltrated dentin. This study tested the hypothesis that interfacial degradation of resin-dentin bonds may be prevented or delayed by the application of chlorhexidine (CHX), a matrix metalloproteinase inhibitor, to dentin after phosphoric acid-etching. Contralateral pairs of resin-bonded Class I restorations in non-carious third molars were kept under intra-oral function for 14 months. Preservation of resin-dentin bonds was assessed by microtensile bond strength tests and TEM examination. In vivo bond strength remained stable in the CHX-treated specimens, while bond strength decreased significantly in control teeth. Resin-infiltrated dentin in CHX-treated specimens exhibited normal structural integrity of the collagen network. Conversely, progressive disintegration of the fibrillar network was identified in control specimens. Auto-degradation of collagen matrices can occur in resin-infiltrated dentin, but may be prevented by the application of a synthetic protease inhibitor, such as chlorhexidine.
The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that... more The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2&amp;amp;amp;amp;amp;amp;amp;amp;#39; of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.
Uploads
Papers by Leo Tjäderhane