Endogenous Enzymatic Activity in Dentin Treated with a Chitosan Primer

The aim of this study was to evaluate the effect of different concentrations of chitosan polymer on dentinal enzymatic activity by means of gelatin and in situ zymography. Human dentin was frozen and ground in a miller. Dentin powder aliquots were demineralized with phosphoric acid and treated with three different concentrations of lyophilized chitosan polymer (1, 0.5 and 0.1 wt%) dissolved in distilled water. Dentin proteins were extracted from each experimental group and electrophoresed under non-reducing conditions in 10% SDS-PAGE containing fluorescein-labeled gelatin. After 48 h in the incubation buffer at 37 °C, proteolytic activity was registered under long-wave UV light scanner and quantified by using Image J software. Furthermore, additional teeth (n = 4) were prepared for the in situ zymographic analysis in unrestored as well as restored dentin pretreated with the same chitosan primers. The registered enzymatic activity was directly proportional to the chitosan concentration and higher in the restored dentin groups (p < 0.05), except for the 0.1% chitosan primer. Chitosan 0.1% only showed faint expression of enzymatic activity compared to 1% and 0.5% concentrations. Chitosan 0.1% dissolved in water can produce significant reduction in MMPs activity and could possibly contribute to bond strength preservation over time.

Zymography-types, advantages, uses and troubleshooting - A detailed review

There are many methods for the study and quantification of enzymes in the body systems. Off-late, the method of zymography has assumed immense importance in diagnostic medicine. It is a method where enzymes are visualized using the substrate conversion technique. The product of reaction appears, or the substrate disappears and the mechanisms specific for the recognition of this measure the biochemical reaction. This procedure has many advantages, the most important ones being providing both qualitative and measurable data, such as molecular zymography methods for visualising hydrolytic enzymes and differentiating among whole molecules, degradation and complexes. There are three main types of zymography, namely- In-Gel zymography (IGZ), In-Situ zymography (ISZ) and In-vivo zymography (IVZ). There are many variations of this technique like transfer 2D and reverse zymography. This method is mainly used to study the expression of matrix metalloproteinases. Alternate to the conventional zymography techniques have been suggested, like usage of the new Ponceau S staining protocol that provides significant benefits in terms of assay usability and cost reduction and is comparatively faster and easier. Mild alterations in the actual procedure of zymography can take care of minor issues arising during the procedure.

Endogenous Enzymatic Activity of Primary and Permanent Dentine

Matrix metalloproteinases (MMPs) play an important role in tooth development and influence caries development and hybrid layer degradation. Literature is scant on the differences in the activity of MMPs between primary and permanent dentine. Accordingly, the aim of the present study was to investigate endogenous gelatinolytic activity in primary and permanent dentine. Separate batches of dentine powder were obtained from intact human primary and permanent molars (n = 6). Each batch was divided in two subgroups: (1) mineralised; and (2) demineralised with 10% H3PO4. After protein extraction, gelatine zymography was performed. Furthermore, in situ zymography was performed on dentine sections of the same groups (n = 3). The slices were polished, covered with fluorescein-conjugated gelatine and evaluated using a confocal microscope. In situ zymography data were analysed using two-way analysis of variance and post hoc Holm–Šidák statistics (α = 0.05). Primary dentine showed poorly defined bands in the zymograms that vaguely corresponded to the pro-form and active form of MMP-2 and the pro-form of MMP-9. In permanent dentine, demineralised powder demonstrated stronger gelatinolytic activity than mineralised powder. In situ zymography identified stronger enzymatic activity in primary etched dentine (p < 0.05). Stronger enzymatic activity recorded in primary dentine may be related to the differences in morphology and composition between primary and permanent dentine.

Dentin Cross-linking Effect of Carbodiimide After 5 Years

Carbodiimide (EDC)–based dentin primers preserve hybrid layer (HL) integrity. However, aging >1 y has not been investigated. The present study examined whether the cross-linking effect of EDC was reflected in dentin bond strength, endogenous enzymatic activity, and the chemical profile of the HL after 5-y aging in artificial saliva. Noncarious human third molars ( N = 42) were cut to expose middle/deep coronal dentin and treated as follows: group 1, dentin etched with 35% H3PO4, pretreated with a 0.3M aqueous EDC primer for 1 min and restored with XP Bond (Dentsply Sirona); group 2, as in group 1 but without EDC pretreatment; group 3, Clearfil SE Bond (Kuraray-Noritake) primer applied to dentin surface, followed by EDC pretreatment as in group 1 and application of bond; group 4, as in group 3 without EDC pretreatment. After composite buildup, the specimens were cut into sticks or slabs, depending on the experiment. All tests were performed at baseline (T0) and after 5 y of aging (T5) in artificial saliva at 37 °C. Microtensile bond strength (µTBS) was tested at a crosshead speed of 1 mm/min until failure. Endogenous enzymatic activity was investigated with in situ zymography. The chemical profile of HL was determined via Raman spectroscopy. Three-way analysis of variance and post hoc Tukey test were used to analyze µTBS and in situ zymography data (α = 0.05). EDC pretreatment and aging significantly influenced µTBS and in situ zymography results ( P < 0.05). Higher bond strength and lower gelatinolytic activity were identified in the EDC-treated groups at T5 ( P < 0.05), especially in the etch-and-rinse groups. Raman spectra revealed less defined amide III peaks in control specimens at T5. The EDC cross-linking effect persisted in the HL for 5 y in terms of bond strength, collagen structure preservation, and dentinal enzyme silencing.

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

Aim. This study sets out to evaluate the antiproteolytic activity of copaiba oil-based emulsion at the resin/dentin adhesive interface union formed with conventional and self-etching adhesives systems. Methods. At in situ zymography, 30 teeth were sectioned 2 mm below the enamel-dentin junction; a smear layer was standardized and subdivided into four groups. Gelatin conjugated with fluorescein was used and taken to the fluorescence microscope for evaluation. In cytotoxicity, the Trypan Blue method was used at four different time points. The tested groups were (G1) control with distilled water; (G2) 2% chlorhexidine (CLX); (G3) emulsion based on copaiba oil (EC) 10% + X; (G4) 10% EC + Y; and (G5) EC 10% alkaline. The zymographic assay used the same groups described, but in 30 seconds and 10 and 20 minutes. HT1080 cells were incubated and submitted to electrophoresis. The gel was analyzed using ImageJ software. Mann–Whitney and Kruskal–Wallis tests were used in the statistical analysis ( p < 0.05 ). Results. ECs showed higher cell viability in the cytotoxicity test and showed a significant difference in 10 and 20 minutes. In the zymographic assay, alkaline EC reduced 67% of MMP-2 activity and 44% of MMP-9 compared to 2% chlorhexidine. At in situ zymography in qualitative evaluation, all groups tested showed inhibition of activity in metalloproteinases. Conclusion. EC showed activity in the inhibition of metalloproteinases in vitro and in situ, especially the alkaline one. The survey shows the possibility of using ECs, a product from Amazonian biodiversity, as a biomodifier in dentistry.

Skin barrier lipid enzyme activity in Netherton patients is associated with protease activity and ceramide abnormalities

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE ( P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls ( P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage ( P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls ( P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.