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Use of methacrylate-modified chitosan to increase the durability of dentin bonding systems Marina Diolosà, Ivan Donati, Gianluca Turco, Milena Cadenaro, Roberto Di Lenarda, Lorenzo Breschi, and Sergio Paoletti Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5014124 • Publication Date (Web): 27 Oct 2014 Downloaded from http://pubs.acs.org on November 9, 2014

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Use of methacrylate-modified chitosan to increase the durability of dentin bonding systems

Marina Diolosàa, Ivan Donatib,*, Gianluca Turcoa, Milena Cadenaroa, Roberto Di Lenardaa, Lorenzo Breschia,c, Sergio Paolettib

a

Department of Medical, Surgical and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, 34129 Trieste, Italy. bDepartment of Life Sciences, University of Trieste, Via Licio Giorgieri 5, 34127 Trieste, Italy. c Present permanent address:

Department of Biomedical and Neuromotor Sciences, University of Bologna, Via San Vitale 59, 40125 Bologna, Italy.

* Corresponding author e-mail: [email protected] tel: +39 040 558 8733

KEYWORDS: Dentine bonding systems; Chitosan; Teeth restoration; Methacrylates; Adhesion.

1 ACS Paragon Plus Environment

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Abstract This study aimed at investigating the effect of a methacrylate-modified chitosan on the durability of adhesive interfaces to improve the clinical performance of dental restorations. Chitosan was modified with methacrylic acid (Chit-MA70) on 16 % of the amino groups. Viscosity, rheology and 1H-NMR spectroscopy were performed to characterise the modified polysaccharide. Chit-MA70 was blended into a primer of an “etch&rinse” experimental adhesive system and tested on human teeth. The presence of methacrylate moieties and of residual positive charges on the polysaccharide chain allowed Chit-MA70 to covalently bind to the restorative material and electrostatically interact with demineralised dentin. The Chit-MA70 containing adhesive system showed values of the immediate bond strength (26.0 ± 8.7 MPa) comparable to the control adhesive system (25.5 ± 8.7 MPa). However, it was shown that upon performing mechanical thermo-cycling treatment of the dental restoration on human teeth, the adhesive with the methacrylate-modified chitosan, in variance with the control adhesive, did not show any decrease in the bond strength (28.4 ± 8.8 MPa). The modified chitosan is proposed as a component of the etch and rinse adhesive system to efficiently improve the durability of dental restorations.


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Introduction Dental caries has historically been considered the most important component of the global oral disease burden. Dental caries is still a major public health problem in most advanced countries as the disease affects 60-90% of school-aged children, and affects nearly 100% of the adult population in the majority of the countries.1 Dentine bonding systems and resin composites are the most common materials used to restore teeth. The adhesive systems interact with the dental tissues using two different strategies: “etch-and-rinse” or “self-etch” technique.2 The current classification of the adhesive systems is based on the number of steps needed for their application.3 Etch-and-rinse adhesives rely mostly on micromechanical interlocking between resin monomers that infiltrate the demineralised dentine surface (created by preliminary acid etching) forming the so-called “hybrid layer”. Self-etch adhesive systems contain acidic monomers that simultaneously demineralise and infiltrate dentine, thus they do not require a separate step to etch substrates in order to form the hybrid layer. The stability of the bonded interface over time is strictly related to the creation of a stable and homogenous hybrid layer, where there is effective coupling of the monomers within the infiltrated substrate.2 The average duration of composite resin restorations is limited to about 6 years.4 Hence, innovative strategies to overcome this limitation are continuously sought. To this end, specific functional monomers have been proposed and added to dental adhesives to influence their mechanical performance.5-8 As an example, 4methylacryloxyethyl trimellitate (4-MET) has been used in orthopaedic and prosthetic dentistry for its adhesive durability to dentine and good biocompatibility.5-7 Similarly 10-MDP has been frequently advocated to stabilise the hybrid layer due to its ability to react with residual hydroxyapatite crystals.8


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Natural polysaccharides have been proposed as biomaterials for cell encapsulation9-11 and proliferation12 and for biomaterials coating.13 Specifically, chitosan is a collective name to describe a family of linear polysaccharides composed of β-1→4 linked Dglucosamine, with some residual interspersed N-acetyl-D-glucosamine. Chitosan is the only natural polycationic polysaccharide and, considering that the pKa of the amino groups is approx. 6.6 at 25 °C and at physiological ionic strength it is soluble in (slightly) acidic medium.14 Chitosan is available in accordance with GLP and GMP specifications that, combined with its biocompatibility and biodegradability,15 make it a

very

appealing

polysaccharide

for

tissue

engineering

and

biomedical

applications.16,17 Indeed, chitosan has been proposed for different medical applications such as topical ocular applications,18 implantation,19,20 injection21 and drug delivery.22 Chitosan is also bio-adhesive, a property which determines an increased retention at the site of application.23 In the field of dental applications, chitosan has already been introduced in a commercial dental adhesive to exploit its antibacterial properties.24 Chitosan is a very versatile polymer that can be readily modified to endow it with peculiar physical-chemical and/or biological properties. The chemical linkage of methacrylate moieties onto the chitosan chain was recently exploited to prepare rechargeable antimicrobial coatings on polyurethane.25 In addition, a photocrosslinkable hydrogel based on chitosan modified with methacrylic acid incorporating pro-neural rat interferon γ was shown to promote the differentiation of adult neural stem/progenitor cells into neurons.26 In the present work, chitosan was modified with methacrylate moieties and a physical-chemical characterisation was carried out. The use of chitosan as a component of the adhesive system is not new. Elsaka et al.24 previously evaluated the use of chitosan blended within the adhesive systems due to its antibacterial properties.


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However, these authors reported a significantly decrease of tensile bonding strength at the dentine interface upon increasing the concentration of chitosan. We speculated that the modified chitosan is able to covalently bind to the restorative resin and, due to the presence of residual positive charges on the polysaccharide, to electrostatically interact with the demineralised dentine. Chitosan bearing methacrylate groups was blended within the primer of a three-step etch & rinse experimental dentine bonding system to assay its bonding ability to dentine. Additionally, bonded interfaces were challenged with a chewing simulation associated with mechanical and thermo-cycling to assay their stability over time. The introduction of chitosan-modified monomers within the primer of an experimental three-step etch-and-rinse adhesive, improved 1) immediate bond strength (T0 µTBS); 2) stability of the hybrid layer after simulated chewing and thermo-cycling (TCS µTBS).

Materials and Methods Chitosan was purchased from Sigma-Aldrich (St. Louis, LO) and purified as reported elsewhere.27 The relative molar mass (“molecular weight”), MW, as measured by intrinsic viscosity,28 was found to be approx. 540,000 and the residual degree of acetylation, as determined by 1H-NMR, was 17 %.29 Lysozyme from chicken egg white (40,000 units/mg protein), methacrylic acid, N-(3-dimethylaminopropyl)-N'ethylcarbodiimide hydrochloride (EDC), N-hydroxy Succinimmide (NHS), Bisphenol A glycerolate dimethacrylate (BisGMA), Triethylene glycol dimethacrylate (TEGDMA),

Ethyl-4-dimethylaminobenzoate

(EDMAB),

Diphenyl-(2,4,6-


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trimethylbenzoyl)-phosphine oxide (TPO), camphorquinone (CQ) were purchased from Sigma-Aldrich (St. Louis, LO).

Chitosan depolymerization Chitosan (1 g) was dissolved in deionised water (100 mL) and it was added of HCl 1 M (7.5 mL) and of NaNO2 10 mg/mL (1.5 mL). The solution was vigorously stirred for 50 minutes and reduced by means of NaBH4.30,31 The pH of the solution was raised to approximately 5.5 with aqueous NaOH, the solution was dialysed against NaCl (0.1 M, 2 shifts) and deionised water until the conductivity was below 4 µS at 4°C, the pH was adjusted with HCl and the solution freeze-dried to obtain the hydrochloride salt of chitosan. The MW value, as determined by intrinsic viscosity measurements,28 was found to be 72000.

Modification of chitosan with methacrylic acid (Chit-MA) Chitosan (660 mg) was dissolved in aqueous MES 0.05 M (250 mL, pH 5.5). Methacrylic acid was added at different ratio [Methacrylic acid]/[chitosan]ru followed by the addition of NHS and EDC ([EDC]/[methacrylic acid] = 1.5 and [NHS]/[EDC]=1). [chitosan]ru indicates the concentration of chitosan repeating units. The solution was stirred for 24 hours, dialysed against NaHCO3 (0.05 M, 3 shifts), aqueous HCl (0.001 M, 2 shifts), aqueous NaCl (0.1 M, 4 shifts), deionised water until the conductivity was below 4 µS at 4 °C and finally freeze-dried. The degree of substitution with methacrylic moieties was determined by 1H-NMR analyses.

Viscosity measurements


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Intrinsic viscosity ([η]) was determined by analysing the polymer concentration dependence of the reduced specific viscosity (ηsp/c) and of the reduced logarithm of the relative viscosity (ln(ηrel)/c) according to the Huggins (eq. 1) and Kraemer (eq. 2) equations, respectively:

η sp

= [η ] + k ' [η ] c

eq. 1

ln (η rel ) 2 = [η ] − k ' ' [η ] c c

eq. 2

c

2

Where k' and k'' are the Huggins and Kraemer constants, respectively. The buffer used was aqueous 0.02 M HAc/NaAc, pH = 4.5 in the presence of NaCl 0.107 M.28 The polymer solutions and the solvent were filtered through 0.8 µm Millipore filters prior to their use. Measurements were performed at 20 °C. Viscosity-average MW values were calculated according to the Mark-Houwink-Sakurada equation, with parameters taken from reference.28

1

H-NMR spectroscopy

1

H-NMR spectra were recorded at 85 °C with a JEOL 270 NMR spectrometer (6.34

T) operating at 270 MHz for proton. The chemical shifts were expressed in ppm downfield from signal for 3-(trimethylsilyl)propanesulfonate.

Scanning Electron Microscopy (SEM) Specimens were analysed using a Quanta 250 Scanning Electron microscope (FEI,

Oregon, USA) visualised in backscattered scanning detection mode at 2500 x after sputter coating with an ultrathin layer of carbon. The working distance was 10 mm and the accelerating voltage was 1kV.


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Attenuated Total Reflectance (ATR) InfraRed (IR) spectroscopy The FTIR-ATR spectra were measured using a Varian FT-660 spectrometer equipped with a diamond crystal GladiATR accessory (Pike Technologies).

Degradation with lysozyme Degradation of native chitosan and of Chit-MA by means of lysozyme was followed by measuring the time dependence of the reduced capillary viscosity (ηsp/c) at 20 °C by means of a Schott-Geräte AVS/G automatic measuring apparatus and an Ubbelohde type viscometer. 12 mL of a 0.8 g/L solution of Chit-MA or chitosan in 0.02 M NaAc/HAc added of NaCl 0.2 M at pH 4.5 were added of 0.5 mL of lysozyme (0.5 mg/mL) dissolved in the same solvent. Prior to measurements, all solutions were filtered through 0.8 µm Millipore filters.

Primer preparation Chit-MA70 (120 mg) was dissolved in aqueous MES buffer (50mM, 6 mL, pH 5.5). The solution was added of HEMA (3.6 mL) and of ethanol (2.4 mL). A control adhesive system was prepared through the same procedure but omitting the presence of Chit-MA70.

R2 resin preparation An experimental resin (R2), similar to the non-solvated hydrophobic resin contained in different commercial bonding agent of three step etch-and-rinse and two-step selfadhesive systems was prepared in accordance with Cadenaro et al..32,33 In present study, R2 was modified adding a second photo-initiator, namely 2,4,6trimethylbenzoyl-diphenylphosphine oxide (TPO), to improve the polymerisation


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kinetic of the hydrophilic moieties blended within its formulation. Final composition of R2 was: BisGMA 70%wt; TEGDMA 28%wt; EDMAB 0.5%wt; TPO 0.5%wt; CQ 0.25%wt. Stock mixtures were prepared by incorporating all components in dark bottles and by stirring them for 2 min by means of a vortex mixer (Vortex Classic, Velp Scientifica, Milano, Italy).

Human teeth specimen preparation Twenty recently extracted, non-carious human third molars were selected, stored in 0.5% chloramine-T solution at 4°C, and used within one month of extraction.34 The occlusal third of the molar crowns and the roots were removed perpendicular to the long axis of each tooth by means of a water-cooled slow speed diamond blade (Isomet 5000, Buehler Ltd, Lake Bluff, IL, USA) to obtain a 4 mm high dentinal section. The exposed dentine surfaces were polished with 180-grit wet silicon carbide abrasive paper to create a standardised smear-layer.35 The dentine disks were then randomly and equally assigned to the 2 groups (N=10): 1) Chit-MA70-containing adhesive system; 2) control formulation without Chit-MA70. The exposed dentine surface was etched with 35% phosphoric acid for 15 s then rinsed with water, gently air-dried in accordance with the wet-bonding technique. The Chit-MA70-containing primer was applied for 20 s and gently air dried to obtain a shiny surface and then R2 resin was applied for 20 s, gently air dried and polymerised for 40 s with a LED lamp (Valo, Ultradent Product Inc. South Jordan, UT, USA; tip distance of 1 mm). The irradiance level of the light was monitored periodically with a radiometer (Demetron 910726, Kerr Corporation. Danbury, CT, USA) to ensure an output of at least ≥ 600 mW/cm2. A 2 mm increment of resin composite (Filtek Z250, 3M ESPE, St Paul, MN, USA) was incrementally layered on the adhesive surface to create a resin-dentine build-up and each increment was light-cured for 20 s. 9 ACS Paragon Plus Environment

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Specimens of the control group were similarly prepared using the primer without Chit-MA70.

Ageing process Half of the bonded specimens of each group (N=5) was randomly assigned to thermomechanical loading to simulate in vitro ageing of adhesive interface. To simulate the clinical situation, the ageing was performed by means of thermo-mechanical loading of the specimens with a CS-4.4 Chewing Simulator coupled with a Thermo Cycling device (SD Mechatronik GmbH, Germany). Specimens selected for occlusal loading were partially embedded in Protemp 4 (Temporisation Material, lot: C 434261, exp. date: 2015-05, 3M ESPE) in a primary Polytetrafluoroethylene (PTFE) mould (diameter of 15 mm). To ensure a perfect parallelism between the resin composite and the aluminium antagonist of CS, specimens were gently pressed with the CS device before Protemp complete hardening. Periodontal ligament simulation was obtained with a 2.5 mm thick layer of Even Express 2 (Impression Material, lot: F 546236, exp. date: 2015-06, 3M ESPE) surrounding the standardised roots (formed by Protemp including natural roots). To simulate 5 year of in vivo service in the oral cavity the aluminium antagonists imprinted an occlusal load of 50N with a frequency of 1Hz and a downward speed of 16mm/s for 1200000 cycles. Simultaneously, specimens underwent 6000 thermal cycles, 5-55°C, of 1 min each temperature with artificial saliva. The test duration was approximately 2 weeks (TCS).36-39 Static conditions (T0 - no thermo-mechanical stresses) were considered as baseline. T0 specimens underwent static storage in artificial saliva at 37°C for 24h.


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MicroTensile Bond Strength test (µTBS) At the two time points, T0 and TCS, resin-dentine build-ups were sectioned perpendicular to the bonded surface using a low-speed diamond saw (Isomet 5000, Buehler Ltd, Lake Bluff, IL, USA) under water irrigation in according with nontrimming technique for microtensile bond strength testing to obtain specimens of 0.9 x 0.9 x 8.0 mm.40 The dimensions of each specimen were individually measured with a digital calliper (± 0.01mm) and recorded for subsequent bond strength calculation. Sectioned sticks were stored for 24 h in artificial saliva at 37°C,41 then glued with cyanoacrylate (Zapit, Dental Ventures of America Inc. Lewis Ct. Corona, CA) to the two free-sliding parts of a microtensile testing machine. The specimens were stressed until failure under tension at a crosshead speed of 1 mm/min (Bisco Inc. Schaumburg, IL, USA). The microtensile bond strength values were then converted into unit of stress (MPa).

Interfacial Nanoleakage Analysis Eight additional teeth were used to evaluate interfacial nanoleakage expression. Disks of middle/deep dentine were prepared, bonded and aged similarly to the specimens submitted microtensile bond strength test. The resin-bonding build-ups were then cut perpendicular to the bonded surface to obtain 1 mm thick specimens. Slabs were immersed in a 50% aqueous ammoniacal silver nitrate (AgNO3) solution in presence of laboratory light for 24 h. Specimens were then photo-developed for 8 h to reduce silver ions into metallic silver grains within voids along the bonded interface and then polished using wet 600, 1200, 2000grit silicon carbide papers.42 Resin-dentine interfaces were observed under optical


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light microscope (Leica DMR; Leica, Wetzlar, Germany) and scanning electron microscopy operating with mixed secondary/backscattered electron mode at 10 kV. The amount of silver nitrate deposit was evaluated and scored by two investigators using the method of Saboia et al..43 Interfacial nanoleakage was scored based on the percentage of the adhesive surface showing AgNO3 deposition: 0, no nanoleakage; 1, nanoleakage on 75% of the adhesive surface. Intra-examiner reliability was assessed using the kappa (κ) test.

Statistical Analysis Microtensile bond strength test data were evaluated using commercial software (SPSS, version 20.0 for Windows; SPSS, Chicago, IL, USA). Values were normally distributed and data were analysed with one-way analysis of variance (ANOVA) and Tukey's post hoc multiple comparison tests. A p-value 75% of the adhesive joint) observed at 100x magnification using optical microscope.

To assess the ability of the primer containing Chit-MA70 to increase the bonding to dentine, human teeth were subjected to two different ageing methods, namely dynamic storage with a chewing simulator coupled with a thermal treatment (CS) or a static storage in artificial saliva for the same time.51 The specimens were analysed performing microtensile bond strength tests (µTBS) after static (T0) and dynamic storage (Tcs). The results of µTBS are reported in Figure 4c. After CS, the bond strength of adhesive interfaces created with the Chit-MA70-containing primer did not show any significant decrease in the bond strength (Chit-MA70 - T0 µTBS = 26.0 ± 8.7 MPa; Tcs µTBS = 28.4 ± 8.8 MPa), while the control specimens (i.e. the same formulation without the addition of Chit-MA70), showed a reduction of 30 % in the bond strength with respect to the values of the static storage (Control - T0 µTBS = 25.5 ± 8.7 MPa MPa; Tcs µTBS = 18.0 ± 6.0 MPa). Conversely, no significant difference in bond strength was found between the Chit-MA70 containing primer and 20 ACS Paragon Plus Environment

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the control after storage in static conditions (26.0 ± 8.7 MPa and 25.5 ± 8.7 MPa respectively). These data suggest that the presence of Chit-MA70, by chemically binding the R2 adhesive through covalent links and by physically binding the organic part of the dentine through electrostatic interactions, increases stability of the bond and stabilises the hybrid layer. Figure 5 presents the extent of AgNO3 deposition (interfacial nanoleakage expression) along the bonded interface for the restoration in the presence of Chit-MA70 containing primer and of the control primer. Adhesive interfaces treated with ChitMA70 containing primer showed lower interfacial nanoleakage expression after either chewing simulation (CS) or static storage in artificial saliva. Conversely, adhesive interfaces created with control primer demonstrated a significant increase in interfacial nanoleakage expression (irrespective of ageing). The increased interfacial nanoleakage in etch & rinse adhesive systems is due to the degradations of collagen fibrils and the hydrolysis of resin from interfibrillar spaces within the hybrid layer. These processes weaken the strength and durability of resin–dentine bond.52 The analysis of interfacial nanoleakage expression of the bonded interfaces showed that, when Chit-MA70 was included in the primer the hybrid layer showed an increased stability.


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Figure 5. Representative images of nanoleakage analysis for each tested group using optical microscope at 100x magnification (A [TCS] and C [T0] for Chit-MA70 containing primer; E [TCS] and G [T0] for the control primer) and backscattered scanning electron microscopy (SEM) at 2500x (B [TCS] and D [T0] for Chit-MA70 containing Primer; Group 2= F [TCS] and H [T0] for the control Primer).

Conclusions An efficient adhesive system is challenged to establish interactions with both the hydrophobic layer (the resins of the restoration material) and the hydrophilic counterpart (demineralised dentine). Nowadays, the durability of the adhesive system represents the major limitation regarding resin-based dental restorations where the hybrid layer between the dentine (hydrophilic component) and the restorative resin (hydrophobic component) is the weakest point. Given the number of caries treated worldwide, the development of novel and better performing dentin bonding systems might have a huge impact by prolonging the durability over time of the dental restoration. The present manuscript aims at testing a primer based on a natural polysaccharide, i.e. chitosan, decorated with methacrylic acid moieties (Chit-MA70) to reach this target. Chit-MA70 shows both hydrophilic and hydrophobic features, coupled with the


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possibility of chemically or physically interacting with both the restorative material and the organic part of the demineralised tooth which makes it a good candidate to enhance the sealing potential and extending dental restoration durability.53 The methacrylate-modified chitosan (Chit-MA70) showed good ability to localise at the dentine-restoration interface with a good infiltration within the dentine tubules. The most interesting finding of the present work was related to the effect of the methacrylate-modified chitosan in the primer used for dental restoration. Indeed, the ageing of the dental restoration with a chewing simulator combined with the thermal treatment (CS group) showed no variation of the bond strength when the methacrylate-modified chitosan was introduced in the adhesive system, at variance with the control dental restoration. Although additional work on the performance of the modified chitosan is needed – and this action is in progress this might be an interesting component to be added to water-based adhesive systems to increase the durability of dental restoration. In addition, the use of chitosan samples with different degrees of substitution with methacrylate moieties and with the presence of spacers between the polysaccharide and the reactive methacrylate group is foreseen for forthcoming work.

Supporting Information Available Detailed composition of the methacrylate modified chitosans used in the present study and the data on the solubility of the modified chitosans in aquesous/ethanol binary solutions are reported in the Supporting Information section. This information is available free of charge via the Internet at http://pubs.acs.org/.


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Acknowledgments This work has been supported by the Italian Ministry of Education (PRIN 2009 – 2009FXT3WL).

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