Effect of ultraviolet A-induced crosslinking on dentin collagen matrix

Highlights

  • For the first time, we evaluated the inhibitory effect of UVA-induced crosslinking on both MMPs and cathepsin-K.

  • UVA-induced crosslinking was tested in the presence/absence of photosensitizer in terms of the loss of dentin mass, degradation by MMPs and cathepsin-K.

  • The results of the study showed that UVA-induced crosslinking significantly inactivated endogenous proteases of dentin.

Abstract

Objectives

The aim of this study was to evaluate the effect of using UVA-induced crosslinking with or without riboflavin as photosensitizers on degradation of dentin matrix by dentin proteases.

Methods

Demineralized dentin specimens (0.4 × 3 × 6 mm 3 , n = 10/group) were subjected to: (RP1), 0.1% riboflavin-5 phosphate/UVA for 1 min; (RP5), 0.1% riboflavin-5 phosphate/UVA for 5 min; (R1), 0.1% riboflavin/UVA for 1 min; (R5), 0.1% riboflavin–UVA for 5 min; (UV1), UVA for 1 min; (UV5), UVA for 5 min. Specimens were incubated in 1 mL zinc and calcium containing media for 1 day and 1 week. An untreated group served as control (CM). After incubation, the loss of dry mass of samples was measured and aliquots of media were analyzed for the release of C-terminal fragment telopeptide (ICTP vs. CTX) of collagen to evaluate for cathepsin K (CA-K) and total matrix metalloproteinase (MMP)-mediated degradation. Data were analyzed using repeated measures ANOVA at α = 0.05.

Results

Although UVA radiation alone reduced dentin degradation, UVA-activated riboflavin or riboflavin-5 phosphate inhibited MMP and CA-K activities more than UVA alone. The effects of crosslinking were more pronounced in 7-day samples; only with CA-K were the effects of crosslinking with or without photosensitizer significantly different from controls in 1-day samples.

Significance

The use of bioactive forms (RP) or longer treatment time did not result with better effect. The use of UVA crosslinking reduces dentin matrix degradation, especially with photosensitizers.

Introduction

In spite of recent improvements in adhesive dentistry, the durability of resin–dentin bonds is still far from optimal due to enzymatic degradation of collagen fibrils in the hybrid layer by endogenous dentin proteases . Among these enzymes, several matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) participating on type I collagen degradation have been reported in both intact and carious dentin . These endogenous proteases are responsible for the host-derived degradation of poorly resin-infiltrated dentin matrix, resulting with the loss of bond strength over time . MMPs are capable of hydrolysis of non-resin infiltrated dentin collagen at neutral pH, whereas cathepsin-K (CA-K: the only cathepsin capable of degrading type I helical collagen) is mainly active under acidic conditions (i.e. pH 5–6) and can also activate MMPs . Phosphoric acid etching of dentin or etching with acidic monomers in resin adhesives leads to activation of both of the classes of proteases .

Improvements in the stiffness of collagen and the inhibition of proteases are two main strategies to protect the integrity of collagen matrix . The effective inhibition of proteases may require targeting both MMPs and CA-K. Crosslinking of dentin collagen improves the stability of bonds by strengthening collagen matrix and by preventing collagen degradation. Recent studies have shown that even brief crosslinker pretreatments are capable of inhibiting endogenous MMP-related dentin degradation over time .

Ultraviolet A induced crosslinking, a new photo-oxidative method using riboflavins has been reported to improve the mechanical properties of organic dentin matrix . Likewise chemical crosslinking, using Ultraviolet A (UVA) radiation is capable of creating covalent intermolecular crosslinking of collagen fibrils by the release of free oxygen radicals that can break weak intermolecular bonds to generate intermolecular covalent bonds . Riboflavin is a biocompatible photosensitizer that can be used to form free radicals when activated by UVA with optimum absorption peaks at 270, 366, 445 nm wavelengths. UVA-induced riboflavin irradiation releases free oxygen radicals that react with collagen amine groups to generate covalent bonds . To be maximally effective, riboflavin in the body fluids should be converted to the phosphorylated bioactive form called riboflavin 5′-phospate (RP) due to the poor solubility of riboflavin in water. However, for local application, there is no need to use the bioactive form, which is more expensive. Additionally, the required riboflavin concentration for photo-activated crosslinking of dentin is similar to its water solubility (0.1–0.5%) .

Previously, this photo-reductive cross-linking has been used in ophthalmology to treat keratoconus by using UVA-irradiated riboflavin to enhance the stiffness of corneal collagen fibrils . Although previous studies have indicated that the treatment of riboflavin by UVA improves the micromechanical properties of dentin and hybrid layers , there are few studies focusing specifically to the inhibition effect of crosslinking on dentin MMPs , and no information is available of the effect on cathepsins. Also, the effect of radiation time and modification of UVA-induced crosslinking with or without riboflavin on degradation of collagen matrix by MMPs and CCs has not been studied. The aim of this study was to evaluate the effect of using UVA-induced crosslinking with or without different riboflavin concentrations, on degradation of dentin matrix by dentin proteases. The null hypotheses were that: (1) treatment of demineralized dentin by UVA with or without riboflavin would not decrease the degradation of collagen matrix by neither CCs nor MMPs over time, and (2) the effect of UVA-induced crosslinking would depend on application time and concentration.

Materials and methods

Preparation of dentin specimens

Seventy extracted sound human third molars removed during a normal treatment under a protocol with patient’s informed consent from University of Oulu Hospital and Health Care Center were used in this study. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth, and were used within three months after extraction. The enamel and superficial dentin were removed parallel to the occlusal surface by using a low-speed diamond saw (Buehler, Lake Bluff, IL, USA) under water-cooling. Dentin specimens (0.4 × 3 × 6 mm 3 ) were sectioned from the mid-coronal dentin. Mineralized dentin specimens were completely demineralized in 10% H 3 PO 4 for 24 h under constant stirring, rinsed in distilled water for 2 h and dried in a vacuum desiccator containing anhydrous silica beads for 72 h to assess their initial dry mass. Specimens were divided into 7 groups ( n = 10). Groups were (1) R1, pretreated with 0.1% riboflavin (Sigma, St. Louis, MO, USA) under UVA light for 1 min; (2) R5, pretreated with 0.1% riboflavin under UVA for 5 min; (3) RP1, pretreated with 0.1% riboflavin-5 phosphate (Fluka, St. Louis, MO, USA) under UVA for 1 min; (4) RP5, pretreated with 0.1% riboflavin-5 phosphate under UVA for 5 min; (5) UV1, exposed UVA light alone for 1 min; (6) UV5, exposed UVA light alone for 5 min. Solutions were prepared in water and kept in light-proof test tubes to avoid any light-activation of riboflavin. The demineralized samples were immersed in 200 μl of 0.1% riboflavin or riboflavin-5-phosphate and exposed to UVA for 1 or 5 min under a UV lamp (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ). Demineralized dentin beams were exposed to UVA light at a distance of 1 cm . They were then turned over and the dose was repeated. UVA has been reported to only penetrate 200 μm into 400 μm thick specimens. Thus, each half of the beam would only receive 1 min or 5 min exposures. Untreated demineralized beams served as control (CM). Groups were incubated in individual labeled polypropylene tubes with calcium and zinc containing incubation media containing 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.02 mM ZnCl 2 , and 0.3 mM NaN 3 , pH: 7.4) for 1 and 7 days at 37 °C in a shaker-water bath (60 cycles/min).

Measurement of dry mass loss overtime

The dry mass for each dentin specimens individually was measured with an analytical microbalance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA) to assess the hydrolysis of total organic matrix of dentin over time. Following demineralization, samples were transferred to individually labeled 96-well plates and placed in the desiccator for 72 h. The initial measurement of dry mass was used as baseline weight of samples. The loss of dry mass was calculated according to the baseline as a percentage after each (1 and 7 days) incubation period. After each measurement, beams were rehydrated for 1 h in distilled water at 4 °C and placed in corresponding polypropylene tubes containing 0.5 mL media. Specimens were rinsed free of salts for 24 h in distilled water at 4 °C after incubation.

Assessment of released collagen telopeptide fragments

To evaluate the specific role of MMPs and CCs in the type I collagen degradation, release of different C-telopeptide fragments was analyzed in the media. MMP activity releases the crosslinked carboxyterminal telopeptide of type I collagen, so-called ICTP, while CA-K among CCs releases a shorter CTX (C-terminal telopeptide of type I collagen) . To analyse the fragments released during incubations of demineralized dentin by proteases, commercial ELISA kits for CTX (Crosslaps ELISA; Immuno Diagnostics System, Denmark) and ICTP (UniQ EIA, Orion Diagnostica, Finland) were used. 50 μl aliquots of media from each beam were used to quantitate solubilized collagen fragments for each time-point. The measurement was performed for aliquots of 10 dentin beams in a spectrometer (Synergy HT, BioTek Inst. Inc., Vermont, USA) at 450 nm absorbance. Collagen degradation as ICTP and CTX telopeptide fragments was calculated in each assay with a standard curve constructed using standards provided by manufacturer.

Statistical analysis

The percentage of dry mass loss after incubation was compared to baseline dry mass for each sample individually before incubation. The loss of dry mass and the quantity of ICTP and CTX release in terms of MMPs and CA-K activity were tested for normality (Kolmogorov–Smirnov test). Since the data were normally distributed, data were analyzed by using repeated measures ANOVA (IBM SPSS v.22, NY, USA and post hoc multiple comparison were tested with Tukey HSD tests (SPSS, IBM Inc., USA). p values of 0.05 were considered to indicate statistical significance.

Materials and methods

Preparation of dentin specimens

Seventy extracted sound human third molars removed during a normal treatment under a protocol with patient’s informed consent from University of Oulu Hospital and Health Care Center were used in this study. The teeth were stored at 4 °C in 0.9% NaCl supplemented with 0.02% sodium azide to prevent bacterial growth, and were used within three months after extraction. The enamel and superficial dentin were removed parallel to the occlusal surface by using a low-speed diamond saw (Buehler, Lake Bluff, IL, USA) under water-cooling. Dentin specimens (0.4 × 3 × 6 mm 3 ) were sectioned from the mid-coronal dentin. Mineralized dentin specimens were completely demineralized in 10% H 3 PO 4 for 24 h under constant stirring, rinsed in distilled water for 2 h and dried in a vacuum desiccator containing anhydrous silica beads for 72 h to assess their initial dry mass. Specimens were divided into 7 groups ( n = 10). Groups were (1) R1, pretreated with 0.1% riboflavin (Sigma, St. Louis, MO, USA) under UVA light for 1 min; (2) R5, pretreated with 0.1% riboflavin under UVA for 5 min; (3) RP1, pretreated with 0.1% riboflavin-5 phosphate (Fluka, St. Louis, MO, USA) under UVA for 1 min; (4) RP5, pretreated with 0.1% riboflavin-5 phosphate under UVA for 5 min; (5) UV1, exposed UVA light alone for 1 min; (6) UV5, exposed UVA light alone for 5 min. Solutions were prepared in water and kept in light-proof test tubes to avoid any light-activation of riboflavin. The demineralized samples were immersed in 200 μl of 0.1% riboflavin or riboflavin-5-phosphate and exposed to UVA for 1 or 5 min under a UV lamp (Philips, Hamburg, Germany; λ = 370 nm at 3 mW/cm 2 ). Demineralized dentin beams were exposed to UVA light at a distance of 1 cm . They were then turned over and the dose was repeated. UVA has been reported to only penetrate 200 μm into 400 μm thick specimens. Thus, each half of the beam would only receive 1 min or 5 min exposures. Untreated demineralized beams served as control (CM). Groups were incubated in individual labeled polypropylene tubes with calcium and zinc containing incubation media containing 5 mM HEPES, 2.5 mM CaCl 2 ·H 2 O, 0.02 mM ZnCl 2 , and 0.3 mM NaN 3 , pH: 7.4) for 1 and 7 days at 37 °C in a shaker-water bath (60 cycles/min).

Measurement of dry mass loss overtime

The dry mass for each dentin specimens individually was measured with an analytical microbalance (XP6 Microbalance, Mettler Toledo, Hightstown, NJ, USA) to assess the hydrolysis of total organic matrix of dentin over time. Following demineralization, samples were transferred to individually labeled 96-well plates and placed in the desiccator for 72 h. The initial measurement of dry mass was used as baseline weight of samples. The loss of dry mass was calculated according to the baseline as a percentage after each (1 and 7 days) incubation period. After each measurement, beams were rehydrated for 1 h in distilled water at 4 °C and placed in corresponding polypropylene tubes containing 0.5 mL media. Specimens were rinsed free of salts for 24 h in distilled water at 4 °C after incubation.

Assessment of released collagen telopeptide fragments

To evaluate the specific role of MMPs and CCs in the type I collagen degradation, release of different C-telopeptide fragments was analyzed in the media. MMP activity releases the crosslinked carboxyterminal telopeptide of type I collagen, so-called ICTP, while CA-K among CCs releases a shorter CTX (C-terminal telopeptide of type I collagen) . To analyse the fragments released during incubations of demineralized dentin by proteases, commercial ELISA kits for CTX (Crosslaps ELISA; Immuno Diagnostics System, Denmark) and ICTP (UniQ EIA, Orion Diagnostica, Finland) were used. 50 μl aliquots of media from each beam were used to quantitate solubilized collagen fragments for each time-point. The measurement was performed for aliquots of 10 dentin beams in a spectrometer (Synergy HT, BioTek Inst. Inc., Vermont, USA) at 450 nm absorbance. Collagen degradation as ICTP and CTX telopeptide fragments was calculated in each assay with a standard curve constructed using standards provided by manufacturer.

Statistical analysis

The percentage of dry mass loss after incubation was compared to baseline dry mass for each sample individually before incubation. The loss of dry mass and the quantity of ICTP and CTX release in terms of MMPs and CA-K activity were tested for normality (Kolmogorov–Smirnov test). Since the data were normally distributed, data were analyzed by using repeated measures ANOVA (IBM SPSS v.22, NY, USA and post hoc multiple comparison were tested with Tukey HSD tests (SPSS, IBM Inc., USA). p values of 0.05 were considered to indicate statistical significance.

Only gold members can continue reading. Log In or Register to continue

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Effect of ultraviolet A-induced crosslinking on dentin collagen matrix
Premium Wordpress Themes by UFO Themes