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 × 2.5 mm 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.
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 6 h, 48.1 ± 8.6 μm and 45.3 ± 6.1 μg for 9 h, 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).
The LM might be a reliable and simplified method to evaluate collagen degradation.
Root caries develops due to collagenolytic degradation of exposed collagen with in the de-mineralized dentin. Inhibition of the collagen degradation is expected to inhibit root caries development . The quantitative analysis of collagen degradation is an important parameter to evaluate caries progression or the efficacy of caries preventive aid . Traditionally, the amount of collagen degradation has been evaluated by hydroxyproline (HPN) assay. This method is sensitive enough to detect even a small amount of collagen degradation, thus the assay has been regarded as a gold standard method to quantify collagen degradation . However this assessment does not provide any visual or dimensional information of the degradated collagen in demineralized dentin lesion. A previous study by Fukuda et al. had conducted a simple approach to measure depth of degraded collagen matrix by polarized light microscopic observation (LM). However the efficacy of such technique has not been established by comparing with the standard technique of collagen degradation assessment.
The aim of this study was to validate the LM technique to evaluate the amount of collagen degradation by comparing with the standard HPN assay technique; furthermore, to evaluate the effect of chlorhexidine (CHX) to prevent collagen degradation by LM technique.
Materials and methods
Preparation of demineralized specimens
30 bovine root dentin blocks, approximately 2 × 3 × 3 mm, were prepared from extracted bovine incisors using a low-speed diamond saw (Isomet, Buehler, IL, USA). The specimens were embedded in self-curing acrylic resin (shade A2, UNIFAST II; GC, Tokyo, Japan) and the dentin surfaces were polished using 2000 grit silicon carbide (SiC) papers (Sankyo, Saitama, Japan). The polished tooth surfaces were covered with acid-resistant nail varnish leaving an uncovered window approximately (1.5 × 2.5 mm) in diameter. The covered areas served as the baseline for sound dentin. Then, the specimens were demineralized with acetate buffer (0.1 mol/L, pH 4.3) for three days at 37 °C (100 mL/10 specimens) to create an incipient lesion.
The collagenase enzyme (type IA, Clostridium histolyticum C-9891, Sigma–Aldrich, Saint Louis, MO, USA) solution contained 50 mmol/L PIPES, 150 mmol/L NaCl and 5 mmol/L CaCl 2 adjusted to pH 6.5. The demineralized specimens were allocated into 3 groups ( n = 10) for different exposure time (6 h, 9 h and 16 h) at 37 °C respectively. Each specimen was placed in 24 wells cell culture plate containing 1.5 mL (0.5 U/mL) collagenase solutions.
Assessment of degraded depth of demineralized collagen matrix LM
After collagenase challenge for each exposure time, the specimens were washed for 5 min and sectioned by a low-speed diamond saw into thin sections 200–220 μm and gently polished using 2000 grit silicon carbide (SiC) papers. Then the sections were placed in between a glass slide and cover slide by adding a droplet of de-ionized water. The images of the specimens were captured by the camera connected to the polarized microscope (SMZ1000, Nikon Corp., Tokyo, Japan). The total area of the degraded collagen and the width of the window were measured by image processing software (ImageJ 1.42). The mean depth of degraded collagen matrix was calculated by dividing the total area of the degraded collagen by its window width as shown in Fig. 1
Following the collagenase challenge for each exposure time, the enzyme solutions were collected for assaying hydroxyproline released from collagen matrix. 60 μL of the solution from each specimen were subjected to hydroxyproline assay using the simplified chloramines-T method . In brief, the solution aliquot was hydrolyzed with 2N NaOH by autoclaving at 120 °C for 20 min. The cloramines-T was added to the hydrolyzate to allow oxidation followed by the addition of Ehrlich’s aldehyde reagent. When chromophore was developed, the absorbance intensity of each solution was read at 550 nm using a spectrophotometer (BIO-RAD–680 micro plate reader, BIO-RAD laboratories, Tokyo, Japan). The trend line was calculated by plotting the optical density of standard hydroxyproline solution with the formula ( y = mx + b ), where y is the instrument response, m represents the slope, and b is an intercept. The actual amount of collagen degradation ( x ) was calculated by the formula; [ x = (OD – b )/ m ], where OD is the corresponding optical density, b is the intercept and m is the slope ( Fig. 2 ).
Anti-collagen degradation efficacy of chlorhexidine gluconate
Demineralized dentin specimens ( N = 10) were treated either with 0.12% CHX (Aldrich, St. Louis, MO, USA) or DW (control) for 1 h at room temperature followed by rinsing with DW, then exposed to the collagenase solution for 16 h as mentioned above. Depth of collagen degradation was compared between CHX and control groups.
The correlation between the LM technique and the HPN assay technique was evaluated by plotting the value of LM against the HPN value. The correlation coefficient, significance, the linearity between 3 incubation time and the efficacy of CHX to prevent collagen degradation were evaluated by statistical software (Sigma Stat Version 16.0, SPSS, Chicago, IL, USA).