Characterization of Riboflavin modified Dentin Collagen Matrix

A. Fawzy, L. Nitisusanta, K. Iqbal, U. Daood, L.T. Beng, and J. Neo J Dent Res 91(11):1049-1054, 2012

Crosslinking Crosslinking is considered a possible approach to the enhancement of mechanical and structural stability and collagen degradation resistance of the demineralized dentin matrix

 The stability (denaturation temperature and resistance against enzymatic degradation) of the fixed tissue is mainly determined by its intrahelical and interhelical crosslinks. In contrast, intermicrofibrillar crosslinks significantly affect the mechanical properties (tissue shrinkage during fixation, tensile strength, strain at break, and ruptured pattern) of the fixed tissue.
J Biomed Mater Res A. 2003 Mar 1;64(3):427-38. Crosslinking of biological tissues using genipin and/or carbodiimide. Sung HW, Chang WH, Ma CY, Lee MH.

Crosslinking can be accomplished by Chemical methods:

Glutaraldehyde, formaldehyde, transglutaminase, carbodiimide, genepin, and proanthocyanidin

Physical methods:
Also called photo-oxidative UV light

Effect of UVA-activated Riboflavin on Dentin Bonding J Dent Res 90(12):1439-1445, 2011

Riboflavin Vitamin B2 Riboflavin is a free-radical-producing agent when photo-activated, with maximum absorption peaks at 270, 366, and 445 nm (Kohlhaas et al., 2006; Spoerl et al., 2007).

 Riboflavin could have potential in adhesive dentistry, owing to its biocompatibility, ease of application, and the ability to be photo-activated by the UVA-blue light spectral range.

Co-enzyme in oxidation reactions and acts as an oxidizing agent because of its ability to accept 2 H+ ions. Reduction of isoalloxazine ring (FAD, FMN oxidized form) yields the reduced forms of the flavoproteins (FMNH2 and FADH2).

Aim of the study

The aim of this study was to investigate the mechanical and chemical variations and collagen degradation resistance associated with crosslinking of the dentin collagen matrix with UVA-activated riboflavin. The null hypothesis was that crosslinking with UVA-activated riboflavin would not significantly affect the mechanical properties, mechanical stability, and hydroxyproline (HYP) liberation of/from the demineralized dentin matrix.

Materials and methods

Sound human molars extracted, for orthodontic reasons, from 70 volunteers (age range, 21-35 yrs) Teeth were stored in 0.5% chloramine-T solution for 2 wks, then in distilled water at 4C until use (Yang et al., 2005) and were used within 1 mo of extraction.

Specimen preparation for crosslinking:

Occlusal enamel removed perpendicular to the long axis of the teeth with a low-speed diamond saw (Buehler, Lake Bluff, IL, USA) under water-cooling until the dentin surfaces, 1 mm below the DEJ, were exposed. Dentin discs, 1 mm thick, cut parallel to the exposed dentin surfaces., which were cut into 4 equal specimens, and each was attached to a disc-shaped metal support. Specimens sequentially wet-ground with 600- to 4,000grit silicon-carbide papers (Carbimet; Buehler, Lake Bluff, IL, USA) and ultrasonicated for 15 min in deionized water.

Riboflavin solution preparation:

riboflavin (RF) solutions (0.1 and 1%) by dissolving riboflavin5-phosphate in distilled water. Solutions were kept in lightproof test tubes to avoid any light- activation of riboflavin
before use.

Application:
Specimens were etched for 15 sec with 37% phosphoric acid and thoroughly rinsed with distilled water for 5 min. The excess water was removed, and the dentin surface was left hydrated and treated with 10 L riboflavin solution (0.1 or 1%) for 2 min.

 Riboflavin was applied in 1 application, left undisturbed for 2 min, gently air-dried for 5 sec, photo-activated with UVA (368 nm) at 7 mW/cm2 for 2 min, and finally gently rinsed with distilled water. The UVA source used (BlueWave, Dymax, Torrington, CT, USA) was placed 10 mm from the dentin surface with a spot-size of 7 mm, such that the whole dentin specimen was fully irradiated with a single UVA irradiation spot. The control specimens were prepared as above but without riboflavin and/ or photo-activation, and only distilled water was used

Evaluation of RB treated dentin by

AFM and SEM Characterization of surface mechanical properties Characterization of bulk mechanical properties Collagen resistance to degradation Micro Raman analysis

AFM and SEM:

The structural changes in the dentin collagen network with collagenase exposure were investigated by AFM and SEM at different time-points. Preparation of collagenase solution: (Zheng et al. 2010)
dissolving 100 mg of collagenase type-I (Clostridiopeptidase A from Clostridium histolyticum, 125 U/mg) in 6 mL of tricine buffer and

3 mL of distilled water.

 Specimens were imaged in liquid mode with AFM and a silicon nitride probe with a nominal tip radius of 10 nm, 18- to 24KHz resonance frequency, and 0.06- to 0.12-N/m springconstant. Tricine buffer was injected into the liquid cell for baseline imaging, followed by withdrawal of the buffer and injection of collagenase solution with continuous feeding for collagenase replenishment for real-time in situ imaging of each specimen at different time-points at a scan rate of 1 Hz. For SEM, specimens were prepared and viewed before and after 24 hrs in collagenase.

Characterization of surface mechanical properties:

Variations in surface nano-mechanical properties were tested (n = 6) at 0, 2, 4, and 8 hrs of exposure to collagenase, with a G200 Nanoindenter System Specimens were fixed to the metal supports and placed in collagenase in Petri dishes, with continuous feeding, until the desired time-point, such that the tested surfaces were facing upward. A pyramid-shaped Berkovich diamond-indenter with a tip radius of 40 nm was used at a constant strain-rate of 0.05/sec with a hold-time of 5 sec at a maximum indentation depth of 70 nm, which corresponded to a load-range of 400-500 N.

 At each specific time-point, specimens were removed from collagenase, gently washed in tricine buffer, and tested in hydrated conditions in the buffer solution, for precise comparison of the readings with the baseline readings in the tricine buffer without exposure to collagenase. Up to 15 indentations were made in the intertubular dentin for each time-point, with lateral spacing not less than 400 nm for each specimen. Youngs modulus (Er) and hardness (H) were calculated

Characterization of bulk mechanical properties:

variations in apparent elastic-modulus (Eappr) and ultimate tensile strength (UTS) after 3 mos in distilled water and 24 hrs in collagenase (n = 17) at 37C were tested and compared with the baseline measurements at 24 hrs in distilled water. Rectangular dentin beams of 0.5 x 1.7 x 6.5 mm (thickness x width x length) were cut and demineralized in 10% phosphoric acid for 5 hrs. Beams for UTS testing were additionally made in an hour-glass shape (0.5 x 0.5 0.1 mm).

 Beams were suspended in the riboflavin solutions, for 2 min, and then each of the opposing sides of each beam was photoactivated separately. Beams were fixed to a testing, and load was applied at 0.5 mm/min crosshead-speed to 3% maximum-strain (Eappr) or to failure (UTS).

Collagen resistance to degradation:

The HYP content in the supernatant was measured at 12, 24, and 48 hrs in collagenase. An HYP assay kit was used Dentin slabs (n = 6) of 4.5 x 3.5 x 0.5 mm were prepared from the mid-coronal dentin and demineralized in 10% phosphoric acid for 5 hrs Aliquots of the supernatants (100 L) were collected and hydrolyzed in 12 N HCl at 120C for 3 hrs. 10-L quantities of hydrolyzed aliquots were transferred to a 96-well plate and evaporated to dryness under vacuum.

 Chloramine-T buffer reagent (100 L) was added and incubated for 5 min at room temperature. a 100-L quantity of DMAB reagent was added and incubated for 90 min at 60C. Absorbance was measured at 560 nm in a spectrophotometer. Standard curves were generated, and HYP content was averaged from quadruplicate measurements.

Micro Raman analysis:

Specimens (n = 5) were prepared as described in microscopic imaging and characterized by micro-Raman with a JY-LabRam HR-800 Raman spectrometer (Horiba Jobin Yvon, Longjumeau, France). Specimens were analyzed before and after crosslinking with photo-activated riboflavin without exposure to collagenase, for characterization of the chemical variations associated with riboflavin crosslinking. Specimens were analyzed with the following micro-Raman parameters: 785-nm excitation wavelength and power < 500 W at a 100X objective.

Statistical Analysis
Data analyzed by two-way ANOVA followed by Tukeys test for repeated-measurement pair-wise comparison within the timepoints and riboflavin concentrations. A p < 0.05 was considered statistically significant

Results:
AFM

SEM

Results:

Discussion:
The null hypothesis was rejected

Mechanism of collagen cross linking by activated riboflavin

Riboflavin exposed to 370nm UV light generates singlet oxygen

This causes cross linking of collagen fibrils by creating covalent bonds between amine groups

Specifically,bridging amine groups (N-H) of glycines of one chain with carbonyl groups (C=O) of hydroxyproline and proline in adjacent chains

Photodynamic Crosslinking of Collagen to Improve Resistance Against Enzymatic Degradation Annie Shrestha, Anil Kishen

 the micro-Raman findings could be attributed to the formation of peptide bonds between dentin and collagen when crosslinked with riboflavin. The stability of the sub-fibrillar triple-helical structure was achieved by the formation of peptide bonds between adjacent collagen chains.

 HYP liberation could result from collagenase mediated collagen degradation, from other dentin enzymes such as cysteine and cathepsins, and from other non-specific collagen-degradation pathways

 lower HYP liberation found with riboflavin crosslinking might indicate the higher collagen content and resistance of the demineralized dentin matrix to bacterial collagenasemediated collagen degradation.

 AFM has an advantage, since it can probe structural variations in the dentin collagen network in the hydrated condition (Fawzy, 2010). However, after extensive surface destruction, due to collagenase effects, SEM could be considered a complementary imaging technique, or as an alternative to AFM.

 nanoindentation testing was used to evaluate surfacemechanical properties since it is more site-specific, Although nano-indentation has high precision, the measurement is limited to only submicron surface depth. However, phosphoric acid demineralizes depths of a few micrometers, leaving behind demineralized collagen network . Therefore, the characterization of bulk-mechanical properties might be more indicative of the penetrative effect of riboflavin crosslinking on the overall mechanical properties of the dentin matrix

 Although other crosslinking agents, such as proanthocyanidins, improved the mechanical stability and collagen resistance to degradation and inhibited the activities of MMPs (Bedran-Russo et al., 2007, 2008; La et al., 2009), the time required to be effective might not be clinically applicable. In addition, crosslinking with glutaraldehyde (Xu and Wang, 2010) is highly effective; however, cytotoxicity is a matter of concern (Huang et al., 1990).

 Higher riboflavin concentrations in the collagen matrix could lead to higher UVA absorption, generating more free-radicals and reactive oxygen species, mainly through the type-I pathway of photosensitized oxidation Type I mechanism involves hydrogen-atom abstraction or electrontransfer between the excited sensitizer and a substrate, yielding free radicals. These radicals can react with oxygen to form an active oxygen species such as the superoxide radical anion. In a Type II mechanism, singlet oxygen is generated via an energy transfer process during a collision of the excited sensitizer with triplet oxygen.
Photosensitized singlet oxygen and its applications Maria C. DeRosa, Robert J. Crutchley Coordination Chemistry Reviews 233/234 (2002) 351/371

 With the increase in dentin thickness and UVA attenuation, a higher concentration of photo-initiator is desired for more efficient crosslinking. However, higher concentrations of riboflavin lead to more yellowish discoloration of dentin, which might adversely affect any subsequent esthetic restoration. In addition, one of the maximum absorption peaks of riboflavin is in the visible blue spectrum. Accordingly, the competition between riboflavin and the photo-activators/initiators of the dentin adhesive system that might affect the polymerization reaction of the resin monomers should be further investigated.

Conclusion
Collagen cross linked with riboflavin had higher degradation resistance to collagenase and enhanced physical properties. The benefits increased with an increase in concentration of riboflavin used.(1%.>0.1%) Avenues to be explored:
riboflavin interaction with adhesive monomer polymerization potential of photo-activation/photo-polymerization in one-step adhesives, optimization of the experimental parameters

Resistance of Corneal RFUVA-Crosslinked Collagens and Small Leucine-rich Proteoglycans to Degradation by Matrix Metalloproteinases Invest. Ophthalmol. Vis. Sci.January 15, 2013 IOVS-1211277 corneal crosslinked collagen type I and type IV are resistant to cleavage by MMP-1, MMP-2, MMP-9 and MMP-13, whereas uncrosslinked collagen I, IV and natively glycosylated SLRPs are susceptible to MMPs. In addition, both crosslinked SLRPs themselves and also crosslinked polymers of SLRPs and collagen appear able to resist degradation. These results suggest that the interactions between SLRPs and collagen caused by RFUVA protect both SLRPs and collagen fibrils from cleavage by MMPs.

Effect of UVA-activated Riboflavin on Dentin Bonding J Dent Res 90(12):1439-1445, 2011

Human dentin was etched with 35% phosphoric acid, treated with 0.1% riboflavin, exposed to UVA for 2 min, and bonded with a two-step etch-and-rinse adhesive. Specimens were subjected to microtensile bond strength tests and pulled to failure after storage for 24 hrs, 6 mos, or 1 yr. Interfacial nanoleakage was evaluated by light and transmission electron microscopy correlative zymographic assays on protein extracts obtained from phosphoric-acid-etched dentin powder with or without riboflavin/UVA treatment and XP Bond to evaluate MMP activity.

Mechanism of Riboflavin Destruction under Light Hyun Jung Kim and David B. Min Department of Food Science and Technology, The Ohio State University, Columbus, OH

Riboflavin produced very unstable and reactive excited diradical triplet riboflavin under light. The reactive and unstable diradical triplet riboflavin produced lumichrome under neutral or acidic pH and lumiflavin under only basic pH by dealkylation. Riboflavin produced electrophilic singlet oxygen under light. Singlet oxygen reacted with electron-rich riboflavin and produced 2,3-butanedione. Sodium azide and ascorbic acid minimized the destruction of riboflavin under light