For clarity, drawing of silica precursor, drug, particles, and schematic diagram of the restoration-tooth interface are not to scale.
Octenidine dihydrochloride (OCT) antimicrobial preserves the resin-dentin interface.
OCT provides long-term inhibition of enzymatic degradation.
OCT provides long-term antimicrobial activity.
Interface OCT level is modulated by a feedback loop mechanism.
Biomaterial testing should use enzymes relevant to pathogenic oral conditions.
Assess the ability of an antimicrobial drug-releasing resin adhesive, containing octenidine dihydrochloride (OCT)-silica co-assembled particles (DSPs), to enhance the biostability and preserve the interfacial fracture toughness (FT) of composite restorations bonded to dentin. Enzyme-catalyzed degradation compromises the dental restoration-tooth interface, increasing cariogenic bacterial infiltration. In addition to bacterial ingress inhibition, antimicrobial-releasing adhesives may exhibit direct interfacial biodegradation inhibition as an additional benefit.
Mini short-rod restoration bonding specimens with total-etch adhesive with/without 10% wt. DSPs were made. Interfacial fracture toughness (FT) was measured as-manufactured or post-incubation in simulated human salivary esterase (SHSE) for up to 6-months. Effect of OCT on SHSE and whole saliva/bacterial enzyme activity was assessed. Release of OCT outside the restoration interface was assessed.
No deleterious effect of DSPs on initial bonding capacity was observed. Aging specimens in SHSE reduced FT of control but not DSP-adhesive-bonded specimens. OCT inhibited SHSE degradation of adhesive monomer and may inhibit endogenous proteases. OCT inhibited bacterial esterase and collagenase. No endogenous collagen breakdown was detected in the present study. OCT increased human saliva degradative esterase activity below its minimum inhibitory concentration towards S. mutans (MIC), but inhibited degradation above MIC. OCT release outside restoration margins was below detection.
DSP-adhesive preserves the restoration bond through a secondary enzyme-inhibitory effect of released OCT, which is virtually confined to the restoration interface microgap. Enzyme activity modulation may produce a positive-to-negative feedback switch, by increasing OCT concentration via biodegradation-triggered release to an effective dose, then subsequently slowing degradation and degradation-triggered release.
Resin composite restorative materials face several host and bacterial challenges to performance, compromising their efficacy as a long-term restorative material. Although aesthetic and mechanical performance of these materials immediately after restoration is quite high, failure due to recurrent caries caused in part by cariogenic bacteria at the restoration margins, or restoration bond fracture, significantly reduces the service life of resin composite restorations [ ]. Salivary and bacterial esterase activity degrades the monomers and resin polymer matrix of these materials [ ]. Low levels of endogenous protease activity from dentinal cathepsins and matrix metalloproteinases (MMPs) may also contribute to the degradation of dentin collagen fibrils [ , ]. The effects of this biodegradation are most acute at the restoration margin where a resin adhesive is used to bond the restoration to exposed type I collagen within the demineralized dentin, producing an interlocking network of collagen and polymer, known as the hybrid layer [ ]. The hydrolysis and resultant degradation and destruction of the hybrid layer results in the reduction of interfacial fracture toughness [ , ]. This degradation also increases microleakage via marginal gap expansion, allowing cariogenic bacteria to penetrate, proliferate and further degrade the interface [ , ]. Degradation by-products increase virulence of cariogenic bacteria [ , ], and human neutrophils contribute to the degradation via their degradative enzymes [ ].
There is a growing body of literature seeking to improve resin-based dental restorative materials through the inclusion of antimicrobial capabilities to combat cariogenic bacteria at the restoration margins [ ]. This may include direct mixing of antimicrobial, mixing antimicrobial-carrying and releasing vehicles, or direct grafting of antimicrobials for surface killing effect or future hydrolytic release [ ]. However the interactions between antimicrobial moieties and the biodegradation of resin materials is rarely investigated, despite evidence that these drugs usually affect esterase and/or proteases activity, and may even prevent biodegradation in a dual-action manner [ , ].
In vivo biodegradation of dental materials can be mimicked using cholesterol esterase (CE) and pseudocholinesterase (PCE) to produce a simulated human salivary esterase (SHSE) media, allowing for more accurate long-term incubation of material and model restoration specimens in vitro in a biochemically dynamic environment [ , ]. Previous studies have elucidated the effect of saliva on restoration bond fracture toughness, mode of failure, and bacterial microleakage at the restoration margin using in vitro studies with SHSE as a valid equivalent to human salivary derived esterases (HSDE) [ ].
We previously developed resin adhesives utilizing antimicrobial drug-silica co-assembled particles (DSPs), which demonstrated high loading of the antimicrobial octenidine dihydrochloride (OCT) and greatly extended drug release period. DSPs were subsequently mixed with a commercial total-etch adhesive at 10% wt. and studied under biochemically realistic conditions via SHSE [ ]. In this study, release of OCT reached a steady state within 15 days via diffusion from resin-embedded DSPs, while the DSPs remain intact to prevent the formation of voids within the resin. In addition to a steady state effective release, OCT release increased following exposure to SHSE. Modeled release dynamics predicted that an effective antibacterial concentration of OCT will be maintained in the restoration marginal gap under worst-case conditions for the patient lifetime (∼30 years), while release of OCT to the broader oral cavity should be minimal due to the low surface area of adhesive exposed at the restoration margin periphery. Furthermore, during steady-state release and significant dilution into a large volume of media, S. mutans biofilm inhibition was maintained at the material surface, demonstrating that an inhibitory concentration of OCT (2 μg mL −1 ) at the resin surface was maintained throughout diffusive release regardless of the sub-inhibitory total incubation media concentration.
The current study aims to assess and compare the interfacial toughness and biochemical stability of resin-composites bonded to human dentin using either DSP-containing, drug free calcined DSP-containing (cDSPs), or stock adhesives. It also assesses the enzyme-modulating capabilities of OCT with respect to the breakdown of the restoration margin, and tests whether released OCT is contained within the interface and not released outside the margins of a realistic restoration geometry. It is hypothesized that the presence of DSPs in the adhesive will enable esterase-modulated release of antimicrobial drug that will preserve the strength of the restoration-tooth bond through drug-enzyme interactions, with no detectable release of the drug outside the interface.
A detailed description of all methods used is included in the supplementary information. Statistical analysis methods are described in each section.
Human teeth and saliva used in this study were obtained according to University of Toronto Human Ethics Protocol #25793. Teeth (3rd molars) were extracted and were handled as before [ ], stored in tap water at −20 °C until use. Cutting of tooth structure was performed with a water-cooled low-speed diamond saw (Buehler Ltd., Lake Bluff, IL). Enamel coronal edges were removed, and coronal dentin slabs were taken from within 2 mm of the cervical line plane before using for various experiments below. Photopolymerization of resins was carried out with a plasma arc device (Sapphire Plus Plasma Arc Curing System, Dent Mat, Santa Maria, CA) for 10 s (adhesive layer) or 20 s (per 1 mm layer of resin composite) at a distance of approximately 1 mm and a minimum intensity of 1730 mW cm −2 as verified by the device’s internal radiometer [ ].
Spherical OCT-DSPs were prepared as described previously, containing 34% wt./50% vol. antimicrobial drug, with an average particle diameter of 424 ± 75 nm [ ]. DSPs were calcined to remove OCT by heating the particles to 600 °C over 2 h followed by a 4 h hold at 600 °C to produce cDSPs with completely open porosity free of OCT. DSP- and cDSP-containing adhesives were prepared to match previous studies and allow direct comparison [ ]: DSPs were added at 10% wt. to the adhesive component of a total-etch adhesive system (Adper Scotchbond Multi-Purpose Adhesive [SBMP], 3M Canada, London, ON), and mixed at 275 RPM for 24 h in darkness. Loss of OCT mass was accounted for when producing cDSP-SBMP utilizing the above procedure to ensure approximately the same volume density of particles within resin (6.6% wt. cDSPs added to SBMP). Adhesives were shielded from light and stored at 4 °C until warming to room temperature before use.
Analysis of restoration-tooth bond fracture toughness
Miniature short-rod (mini-SR) fracture toughness specimens were prepared as described and validated previously to reliably and relevantly assess ex vivo restoration bonding efficacy and degradation (schematic of mini-SR specimen dimensions used in Fig. 1 A) [ , , ]. Details of specimen fabrication are provided in the supplementary information section, using the adhesives prepared in Section 2.1 and a commercial resin composite (Filtek Z250 Universal Restorative, shade A1, 3M Canada, London, ON). Specimen bonding to human dentin was preformed using DSP-SBMP, cDSP-SBMP, or SBMP systems to analyze fracture toughness.
DSP-SBMP and SBMP mini-SR fracture toughness specimens were tested immediately post-synthesis or after 30 or 180 days incubation in simulated human salivary esterase (SHSE) as previously described (N ≥ 6 per experimental group per timepoint) [ , ]. cDSP-SBMP specimens were tested immediately post-synthesis to isolate any potential physiomechanical effect of the novel filler on adhesive bonding performance. SHSE was prepared using cholesterol esterase (CE, Toyobo Co. Ltd., Osaka, Japan) and pseudocholine esterase (PCE, Sigma Aldrich, Mississauga, ON), with average esterase activity matched to that found in human saliva over the 5- to 10-day periods between refreshment or replacement [ , ].
At the end of each respective incubation period, mini-SR specimens were attached to a microtensile tester (Bisco, Richmond, BC). Testing proceeded at 1 mm min −1 in the direction shown in Fig. 1 B. Force at failure (P c ) was recorded and used to calculate interfacial fracture toughness (K Ic ). The effect of friction within the horizontal testing apparatus was equal and negligible (<0.1 N, or approximately 1% of total measured range of fracture toughness).
Statistical Analysis: was performed using ANOVA and least squares fit with a lack of fit test to assess effect significance, and Tukey’s HSD post-hoc analysis to compare groups, with incubation time and material independent variables, and fracture toughness dependant variable (p < 0.05). The O’Brien test was used to assess homogeneity of variance (p < 0.05).
Analysis of OCT release from ex vivo restoration-tooth bonded interfaces
Ex vivo model specimens simulating the gingival margins of proximal or cervical restorations [ , ] were prepared to investigate OCT release outside the restoration margins using a 4 × 4 mm human dentin bonded to composite using DSP-SBMP. Specimens were then incubated at 37 °C for 10 days in 3 mL of PBS or SHSE as in Section 2.2 (N = 10). Incubation media were combined with equal parts methanol to halt esterase activity before analyses via high performance liquid chromatography (HPLC) in combination with UV spectroscopy (281 nm) (Waters, Mississauga, ON) to quantify the mass of OCT released [ , ]. Further details are provided in the supplementary information section.
Effect of OCT on protein adsorption
Effect of OCT on enzyme and salivary protein adsorption to resin surfaces was assessed using a procedure adapted from Zhang et al. [ ]. Disc-shaped photopolymerized SBMP specimens 8 × 0.5 mm were fabricated in a mould and cured 10 s on each side as described above. Specimens were incubated in PBS with 0.2, 2, and 20 μg mL −1 of OCT for 2 h at 37 °C. Identical DSP-SBMP specimens were fabricated and incubated with PBS (0 μg mL −1 OCT) to analyse the effect of surface DSPs and released OCT into a non-OCT-containing buffer. OCT concentrations represent 0.1×, 1×, and 10× the MIC of OCT against S. mutans [ ]. Specimens were then incubated for 2 h in either 4.5 μg mL −1 CE or 1/10 diluted saliva pooled from 4 donors with OCT at 37 °C. The 1/10 dilution of saliva allowed for decreased viscosity and ease handling of whole saliva without further processing which may affect enzyme activity. Adhered protein was removed by sonicating 20 min in 1% sodium dodecyl sulfate (SDS, Sigma Aldrich, Mississauga, ON) in PBS and concentration determined using a bicinchoninic acid total protein assay kit (Sigma Aldrich, Mississauga, ON).
Statistical analyses: Protein adsorption was compared to OCT-free control using Welch’s t-test, and all-group comparisons were made using ANOVA and Tukey’s HSD post-hoc analysis (N = 4 for all groups).
OCT effect on margin degrading enzymes’ measured activity and biodegradation of dentin and resin
Statistical analyses: All results in the below subsections were analysed using ANOVA and Tukey’s HSD multiple comparisons test (p < 0.05, N = 3 unless otherwise specified).
Preparation and incubation of human dentin specimens in OCT incubation media
The effect of OCT on endogenous protease activity and bacteria-derived collagenase was assessed. Coronal dentin from human teeth (N = 3) was collected as described earlier and ground to an approximate particle size of 0.1 mm using an electric blade grinder (Black + Decker) for 20 s. Powdered dentin was then demineralized in 10% phosphoric acid (Fisher Chemical, Ottawa, ON) under constant mixing for 24 h [ ]. Demineralized dentin was then washed 3 times in PBS and 25 mg wet aliquots were suspended in 1 mL of PBS with 0, 0.2, 2, or 20 μg mL −1 OCT and with or without 50 U mL −1 of bacterial collagenase type 1 (from Clostridium histolyticum, Sigma-Aldrich, Oakville, ON, Canada) as a positive control for collagen degradation. Specimens were incubated at 37 °C.
OCT effect on endogenous dentinal MMP activity by fluorometric substrate
Vials were centrifuged for 5 min at 1250 RPM, and supernatant of 0, 0.2, 2, and 20 μg mL −1 OCT-dentin specimens (without bacterial collagenase type 1) were analyzed after 14-days incubation for generic and specific (MMP-1, -2, -8 and -9) MMP-like activity utilizing fluorometric substrates (Sensolyte 520 generic MMP, MMP1, -2, -8 and -9 assay kits, AnaSpec, Fremont, CA) [ ]. 50 μL of supernatant was combined with 50 μL of fluorometric peptide substrate as per manufacturer instructions in a black 96-well plate, and fluorescence was monitored at Ex/Em = 490 nm/520 nm every minute for 1 h (Cytation 5, Bio-Tek, Winooski, VT). Controls of OCT and PBS solutions with and without substrates were utilized for background correction. The resultant fluorescence reading slope was taken as relative MMP-like activity.
OCT Effect on the degradation of human dentinal collagen
At 24 h supernatant from collagenase type 1-containing OCT-dentin specimens were analysed for hydroxyproline (Hyp) content as a marker of bacterial collagenase mediated collagen degradation [ , ] via a commercial amino acid derivatization product and protocol (AccQ-Tag, Waters, Mississauga, ON) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS, Acquity H-class with C18 column, coupled to a Xevo G2-XS Q-ToF MS with electro-spray ionization source, and quantified with QuanLynx analysis software, all from Waters, Mississauga, ON).
After 1, 14, and 36 days of incubation supernatant of specimens without added collagenase type 1 were analysed for Hyp content as before. The supernatant of these 36-day incubated specimens without added bacterial collagenase were also analysed for collagen fragment content as a marker of partial collagen degradation; supernatant was digested via trypsin (Trypsin Gold, Promega, Madison, WI), and analysed using UPLC-MS/MS as before in positive MS E . Results were quantified relatively using Progenesis QI for Proteomics (v4.2, Nonlinear Dynamics, Durham, NC) and searched against the UniProt database [ ].
Two complementary methods to detect release of collagen degradation by-products by endogenous proteases were used to increase the sensitivity of the measurement. Dentinal collagen that has undergone complete digestion and solubilization by protease will release hydroxyproline [ , ]. The method above used can detect quantities of Hyp released as low as 1 ng mL −1 . Collagen fragments in the form of peptides may also be released [ ] either as identifiable by-products of specific protease cleavages, or as detached and solubilized collagen fragments, and are quantifiable by label free analysis via MS/MS. Although the sensitivity of this method is not as high as Hyp derivatization, incomplete collagen digestion not resulting in significant Hyp release could be detected using the latter method.
OCT effect on model and salivary esterase activity
The effect of OCT on stock CE and salivary CE-like esterase activity was assessed. OCT was prepared at 0.2, 2, and 20 μg mL −1 in PBS with either 16 U mL −1 CE, 1/10 diluted whole unstimulated human saliva in PBS pooled from 3 donors as before to analyse human salivary derived esterases (HSDE), or S. mutans bacterial esterase (SMU_118c) in PBS at 0.05 mg mL −1 [ ]. Samples were incubated 24 h at 37 °C. CE-like activity was measured using para-nitrophenyl butyrate (p-NPB) as a colorimetric substrate.
Biodegradation of bisphenol A-glycidyl methacrylate (bisGMA), a universal monomer of adhesive and resin composite, and the primary monomer of SBMP [ ], by CE, HSDE, or SMU_118c was measured with and without OCT. Solutions were prepared and incubated as above, replacing p-NPB with 100 μM bisGMA [ ]. BisGMA-derived biodegradation by-product 2,2-bis[4(2,3-hydroxypropoxy)phenyl]propane (bisHPPP) was quantified via UPLC-MS/MS and photodiode array (as in Section 2.5.3 ) as a marker of biodegradative activity.
Analysis of restoration-tooth bond fracture toughness
Fracture toughness results of mini-SR specimens of composite bonded to human dentin using SBMP or DSP-SBMP are shown in Fig. 1 C. Initial post-fabrication non-aged interfacial fracture toughness values were not statistically different between stock SBMP, DSP-SBMP ( Fig. 1 C). cDSP-SBMP specimens had an immediate fracture toughness of 0.80 ± 0.35 MPa m −1 which was not statistically different from either stock SBMP or DSP-SBMP (Tukey’s HSD, p > 0.05). Incubation time had a statistically significant negative effect on fracture toughness in control SBMP specimens (ANOVA and least squares fit, p < 0.05). Specimens made using DSP-SBMP show no statistically significant change in their fracture toughness values over time (ANOVA and least squares fit, p > 0.05). At 180-days incubation DSP-SBMP specimens had significantly higher fracture toughness values than stock SBMP (Tukey’s HSD, p < 0.05). For the complete ANOVA model, the achieved p-values correspond to an observed power of 0.847.
Analysis of OCT release from ex vivo restoration-tooth bonded interfaces
OCT was not detected by HPLC (detection limit of 4.34 ng mL −1 , corresponding to 26 ng OCT per day) in either PBS or SHSE during the first 10 days of incubation with restoration-dentin specimens utilizing DSP-SBMP adhesive, when release rate of OCT into the interface, and subsequently outside the interface is expected to be the highest [ ].
Effect of OCT on protein adsorption
Protein adsorption results are presented in Fig. 2 . At high 20 μg mL −1 of OCT, CE adsorption on SBMP was significantly inhibited by 28 ± 14% ( Fig. 2 A, Welch’s t-test p = 0.047). DSP-SBMP also showed a statistically significant inhibition of CE adsorption compared to SBMP + 0 μg mL −1 control with a reduction of 35 ± 25% (Welch’s t-test p = 0.040). However no overall effect of OCT or DSPs were seen comparing all groups simultaneously (ANOVA p > 0.05, Tukey’s HSD analysis p > 0.05) No effect on total protein adsorption from saliva is seen at tested concentrations of OCT ( Fig. 2 B, ANOVA p > 0.05, Tukey’s HSD analysis p > 0.05, Welch’s t-test p’s > 0.05).