Highlights
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Electrospraying was successful to obtain chitosan particles encapsulated with and without DCPA.
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Antimicrobial effect was by due attributed to direct contact of the bacteria with exposed with the particles.
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Mechanical properties were not affected reduced by the presence of chitosan or chitosan/DCPA.
Abstract
The incorporation of antimicrobial agents in restorative dental composites has the potential to slow the development of carious lesions.
Objective
The objectives of the present study were to develop experimental composite resins with chitosan or chitosan loaded with dibasic calcium phosphate anhydrous (DCPA) particles and to demonstrate their antimicrobial potential without loss of mechanical properties or biocompatibility.
Methods
Chitosan and chitosan/DCPA particles were synthetized by the electrospray method. Experimental composites were formulated by adding 0, 0.5, or 1.0 wt% particles into a resin matrix along with 60 wt% barium glass. The degree of conversion and mechanical properties were measured after 1 and 90 days of aging in water after photoactivation. Cytotoxicity and genotoxicity were evaluated using fibroblasts from dental pulp in conditioned medium. The antimicrobial activity against Streptococcus mutans was assessed by crystal violet biofilm assay.
Results
The experimental restorative composites were not found to be cytotoxic or genotoxic, with cell viability of 93.1 ± 8.0% (p = 0.328) and 3.0 ± 0.8% micronucleus per group (p = 0.1078), respectively. The antimicrobial results showed that all composites with approximately 20% less biofilm (p < 0.001) relative to the control. No chitosan release was detected from the composites, suggesting direct contact of the bacteria with exposed chitosan particles on the surface was responsible for the observed antimicrobial effect. The addition of the chitosan and chitosan/DCPA submicrometer (<250 nm average diameter) particles to restorative composites did not change the degree of conversion, flexural strength, elastic modulus and fracture toughness compared to the control group after 90 days aging in water.
Significance
It can be concluded that the addition of chitosan or chitosan/DCPA particles in the restorative composites induced antimicrobial activity without compromising the mechanical properties or biocompatibility of the composites.
1
Introduction
People are living longer and retaining more of their natural teeth. One consequence of tooth retention in older adults is an increased risk for development of caries [ ]. Also, recurrent caries at the tooth/restoration interface is prevalent among adults and the elderly, being one of the main reasons for restoration failures [ , ]. To address this issue, experimental bioactive composites have been proposed in order to slow the recurrent decay process.
Chitosan is an emerging natural antimicrobial agent for the dental field [ , ]. Chitosan is derived from chitin which is a large source of sustainable biomaterial. Chitin can be extracted from seafood shells and represents an opportunity to recycle the large amount of seafood waste that is produced globally every year [ ]. The use of chitosan as a bioactive compound is very attractive due to its favorable properties such as low toxicity, high biodegradability, high biocompatibility, and Generally Recognized as Safe (GRAS) status from the US Food and Drug Administration [ ].
The application of chitosan-based biomaterials has shown promising results in tissue regeneration therapies when used in implants coatings [ ], scaffolds [ , ], and drug delivery agents [ ]. The broadening application of chitosan as a bioactive compound has been somewhat limited though due to its low solubility in water, whereby it is only soluble in acidic aqueous solutions [ ].
Chitosan in its powder form has been tested in dental adhesives and composites [ , ], glass ionomer [ ] and resin denture base materials [ ]. The main complication found by adding chitosan powder was a significant reduction in the mechanical properties [ , , ]. Different synthesis techniques have been suggested to overcome this limitation. For example, electrospinning has been used to produce nanofibers [ , ], while electrospray has been shown to be an efficient method to synthesize microspheres [ ].
Furthermore, the electrospray method can produce chitosan microspheres that encapsulate other bioactive compounds [ , ]. Various calcium phosphate based fillers have long been known to remineralize the tooth structure but have also been found to weaken resin based dental composites [ ]. Thus, careful attention must be paid to the balance between bioactive and mechanical properties before such experimental composites can become commercially viable. Electrospray is a possible approach to incorporate calcium phosphate into the resin matrix without jeopardizing its mechanical properties.
Accordingly, in the present work the authors have used the electrospray technique to synthesize novel chitosan microspheres both with and without dibasic calcium phosphate anhydrous (DCPA) with the primary goal of achieving antimicrobial capacity with small additions that do not sacrifice the mechanical properties.
The first objective of this study was to demonstrate the synthesis of novel experimental resin based dental restorative composites containing either submicrometer chitosan particles, or chitosan particles loaded with DCPA. The second objective was to evaluate the chemical, biological and mechanical properties of the experimental materials, and better understand the mechanism of antimicrobial activity. The hypotheses of this study are that the chitosan containing experimental restorative resin-based dental composites are non-toxic, have stable mechanical and chemical properties that comparable with commercially available composites, and also possess antimicrobial properties.
2
Material and method
2.1
Development of experimental composites
Submicrometer particles of chitosan or chitosan loaded with dibasic calcium phosphate (DCPA) were synthetized by the electrospray technique. Chitosan powder (Sigma-Aldrich) with molecular weight of 310−375 kDa and degree of N -deacetylation >75% and dibasic calcium phosphate powder (Sigma-Aldrich) were used to prepare the electrospray solutions. Both powders were in accordance with United States Pharmacopeia (USP) standards. Two different solutions were synthesized by dissolving either (1) 10 mg/mL chitosan or (2) 10 mg/mL chitosan plus 2.5 mg/mL DCPA in 90% acetic acid. The solutions were electrosprayed (TL-01, Long Li Tech., Shenzhen, China) using a flow rate 0.5 mL/h, a voltage of 30 kV, and a distance from the needle to the collector plate of 18 cm. The particles were crosslinked by exposure to glutaraldehyde vapor. This was achieved by sealing liquid glutaraldehyde (25%) in a container with the powder for 16 h at room temperature to allow the vapor to saturate the atmosphere and induce crosslinking. This methodology produced particles with diameters (mean ± standard deviation) of 237 ± 263 nm for chitosan and 216 ± 232 nm for chitosan/DCPA.
Experimental composites were formulated using a 1:1 molar ratio of triethylene glycoldimethacrylate (TEGDMA) and bisphenol A glycidyl methacrylate (BisGMA). Camphorquinone and 2-(dimethylamino) ethyl methacrylate were used as initiators at 1 wt% each and 60 wt% of silanized barium–aluminum–silicate glass (0.7 μm, 5% silane, FGM, Brazil) was added as the reinforcing filler. The submicron particles of chitosan or chitosan loaded with DCPA were mechanically incorporated to the material at 0, 0.5, or 1 wt% under vacuum (SpeedMixer, FlackTek Inc., SC, USA). The resin-based dental composites were handled at the absence of light and removed from the refrigerator for a minimum of 2 h prior to sample preparation. For all subsequent testing, samples were light-cured with an irradiance of 1200 mW/cm 2 using a light curing unit (Radii-Cal, SDI, Australia) as specified below for each specimen geometry. This light curing unit ramps during the first 5 s until an irradiance of 1200 mW/cm² is reached, after which the irradiance stays constant for the remaining polymerization time.
2.2
Biological properties
2.2.1
Cytotoxicity and genotoxicity
After approval by the Ethical Committee in Research from Ibirapuera University (CAAE: 76043917.8.0000.5597) and consent from the patients, human fibroblasts from the dental pulp of teeth with surgical indication were isolated and cultured after surgery in Dulbbecco’s modified Eagle medium (DMEM) with 10% bovine fetal serum. Cytotoxicity test was performed using the conditioned medium technique according the ISO standard 10993 [ ], and genotoxicity was evaluated by counting of micronuclei. For both assays, specimens of 5 mm diameter and 1 mm height were photoactivated for 20 s using a silicon mold and pressed flat with clear strips and a glass slide. The samples were dry stored at 37 °C for 24 h and then immersed in DMEM for 24 h, after which they were immediately tested. For cytotoxicity analysis, 10,000 fibroblasts per well were added and cultured for 24 h in 96 well dishes (n = 3 for each group) in the presence of fresh DMEM (positive control), or the DMEM preconditioned with the composite samples added at concentrations of 100 vol%. Cell viability was assessed by MTT assay and was calculated as the percentage relative to the positive control groups. For genotoxicity, 1 × 10 5 fibroblasts were added per well (n = 3 for each group) in six-well dishes and cultured for 24 h on glass coverslips. The fresh DMEM (positive control) and preconditioned DMEM experimental groups were immediately added to the wells in 100% concentration and maintained for another 24 h. The cells were then fixed by immersing the glass coverslips in 1.5% formaldehyde for 20 min followed by immersion in 20% cold methanol for 20 min. The coverslip was then sealed with Vetashield® mounting medium (Vector Laboratories Inc., Burlingame, CA, USA), which contained DAPI for nuclear staining. The percentage of micronuclei was determined from 100 cells (fibroblasts). The counting was performed in three predetermined microscopic fields (at the two corners and in the center of the coverslip) with a magnification of 400×. All experimental groups were performed in triplicate.
2.2.2
Chitosan release
The Kaiser test was performed to determine if chitosan was released from the experimental dental composite and to help select an appropriate experimental method for testing antimicrobial efficacy. This test used ninhydrin, which is a very sensitive reagent to detect primary amine [ ]. The free N-terminal amine group of the chitosan particles reacts with ninhydrin to produce an intense blue color.
1M ninhydrin solution was prepared by dissolving 4.45 g of ninhydrin crystals (Sigma Aldrich) in 25 mL of deionized water and heating at 80 °C in a water bath for 5 min. Disc shaped specimens were prepared (4.5 mm diameter × 1.0 mm height) using a stainless steel mold. Uncured composite was pressed flat with clear strips and a glass slide, and was then light activated for 20 s (10 s on each side) followed by wet-polishing with 2000/4000-grit silicon carbide paper. Samples were ultrasonically cleaned for 10 min in distilled water and sterilized under UV irradiation for 15 min on each side. Disk specimens (n = 3, each group) were then separated into two groups: (1) tested immediately (2) aged in deionized water for 3 weeks at 37 °C.
For the first group, the Kaiser test was performed by directly adding the experimental composite samples into the 1M ninhydrin solution in ethanol (50% v/v). For the second group, the 1M ninhydrin solution in ethanol (50% v/v) was added to the storage solution. The 0% chitosan control group was used as negative control and the positive control group was prepared by adding 10 μg of chitosan particles into the 1M ninhydrin solution in ethanol (50% v/v). All groups were held in a water bath at 80 °C for 3 h.
2.2.3
Antimicrobial activity
The bacteria inoculum was prepared from a frozen stock using a single bacterial strain of Streptococcus mutans (ATCC 700610). Dehydrated brain heart infusion (BHI) media (Thermo Fisher Scientific Inc., Waltham, MA, USA) was prepared according to manufacturer instructions and the inoculum was prepared directly with fresh broth. The bacteria were grown overnight at 37 °C and 5% CO 2 and diluted to achieve a turbidity equivalent to a 0.5 McFarland standard (OD 600 = 0.5), which is equivalent to 1.5 × 10 8 CFU/mL.
While disk diffusion inhibition halo experiments were also considered in preliminary experiments, based on the Kaiser test results showing no chitosan release ( Fig. 1 ), it was assumed no diffusion could occur and a direct contact test method was chosen to test the antimicrobial efficacy. Disc-shaped specimens (n = 10 for chitosan/n = 16 for chitosan/DCPA group) were prepared following the protocol described for the Kaiser test. For the biofilm formation, 200 μL of bacterial culture was added to 50 mL of fresh BHI with 1% sucrose. The disc specimens were placed in the bottom of a 24 well plate and 2 mL of the bacterial solution was added per well. The samples were then incubated at 37 °C and 5% CO 2 for 24 h.
After 24 h, 2 mL of 0.1 % crystal violet (Sigma Aldrich) was added to each specimen and incubated for 20 min at room temperature. The disc samples were washed three times in deionized water to remove excess stain, air dried, and destained with 2 mL absolute (>99%) ethanol. The biofilm was evaluated by optical density (OD) absorbance using 595 nm wavelength light in a spectrophotometer (UV-1280, Shimadzu, Japan). The background staining was calibrated with absolute ethanol. Antibacterial effectiveness for each experimental composite was calculated as the percentage of the mean OD for the control composite. Biofilm assays were performed in triplicate to ensure repeatability.
2.3
Mechanical properties
2.3.1
Specimen preparation
Unnotched beam specimens (2 mm × 2 mm × 25 mm) were prepared for flexural strength (n = 10 for each group) testing. A stainless-steel rectangular split mold was filled with the restorative material and the uncured computed was pressed flat with clear strips and a glass slide prior to light-curing. Half of the specimens were stored in distilled water at 37 °C for 24 h, while the other half were stored for 90 days.
Notched beam specimens (2 mm × 2.5 mm × 25 mm, n = 5) were made for fracture toughness experiments. A sharp notch was made by inserting a razor blade into a groove on the split mold at mid-point of specimen’s length. The uncured composite was pressed flat with clear strips and a glass slide and photoactivated.
The specimens were light-cured by three adjacent exposures of 20 s. Any excess composite was removed by wet-polishing with 2000/4000-grit silicon carbide paper.
2.3.2
Flexural strength and elastic modulus
Flexural strength was determined using three-point bending with a support span distance, S , of 20 mm in general accordance with ISO standard 4049 [ ]. The specimens were loaded to fracture using a crosshead speed of 0.75 mm/min. Flexural strength was calculated according to:
FS=3PL2bh2
where P is the load at the fracture point, L is the support span distance, b is the specimen width and h the specimen height [ ].
The slope of the linear elastic region in the load-displacement curve was used to determine the modulus of elasticity ( E f ) in bending according to the following equation:
Ef=αL34bh3
where α is the slope of load-displacement curve.
2.3.3
Fracture toughness (K IC ) SEVNB
The notched specimens were tested according to ISO standard 23146 [ ] in a three-point bending test arrangement ( L = 20 mm; roller diameter = 2 mm). Fracture toughness tests were conducted using a universal testing machine (Instron 4501, Instron Corp., Canton, MA) with a crosshead speed of 0.75 mm/min until fracture occurred. K IC was calculated from the peak load at fracture according to the standard stress intensity factor equation for the SEVNB sample geometry:
KIC=PLBW3/2x3aW1/21.99-aW1-aW2.15-3.93aW+2.7aw221+2aW1-aW3/2
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