Bulk fracture is a primary reason for resin-based dental restoration failures.
Self-healing resin contained microcapsules with TEGDMA-DHEPT healing liquid.
A self-healing efficiency of 65% in KIC was obtained with 10–20% microcapsules.
Self-healing resin-based restorations can heal cracks and increase the durability.
Bulk fracture is one of the primary reasons for resin-based dental restoration failures. To date, there has been no report on the use of polymerizable dental monomers with acceptable biocompatibility to develop a resin with substantial self-healing capability. The objectives of this study were to: (1) develop a self-healing resin containing microcapsules with triethylene glycol dimethacrylate (TEGDMA)- N , N -dihydroxyethyl- p -toluidine (DHEPT) healing liquid in poly(urea-formaldehyde) (PUF) shells for the first time, and (2) determine the physical and mechanical properties, self-healing efficiency, and fibroblast cytotoxicity.
Microcapsules of polymerizable TEGDMA-DHEPT in PUF were prepared via an in situ polymerization method. Microcapsules were added into a BisGMA-TEGDMA resin at microcapsule mass fractions of 0%, 5%, 10%, 15% and 20%. A flexural test was used to measure composite strength and elastic modulus. A single edge V-notched beam method was used to measure fracture toughness K IC and self-healing efficiency.
Flexural strength and elastic modulus (mean ± sd; n = 6) of resin containing 5–15% microcapsules were similar to control without microcapsules ( p > 0.1). Adding microcapsules into the resin increased the virgin K IC , which was about 40% higher at 15% microcapsules than that with 0% microcapsules ( p < 0.05). Specimens were fractured and healed, then fractured again to measure the healed K IC . A self-healing efficiency of about 65% in K IC recovery was obtained with 10–20% microcapsules. All specimens with 0–20% microcapsules had fibroblast viability similar to control without resin eluents ( p > 0.1).
Self-healing dental resin containing microcapsules with polymerizable TEGDMA-DHEPT healing liquid in PUF shells were prepared for the first time with excellent self-healing capability. These microcapsules and self-healing resins containing them may be promising for dental restorations to heal cracks/damage and increase durability.
Dental caries is a prevalent problem worldwide with a significant financial burden . Resin composites are popular materials to fill tooth cavities due to their excellent esthetics and direct-filling capabilities . However, the durability of these restorations is limited, with half of all restorations failing in less than 10 years, and with fracture as one of the primary reasons for failure . It was reported that more than 25% of composite replacements were driven by some forms of fracture . Indeed, fracture of restorations was the main reason for failure in composite restorations in large cavities after 5 years of use .
A main reason that composite restorations fail was suggested to be the formation and accumulation of micro-cracks, caused by factors including mastication forces and thermal stresses . Efforts have been made to develop composites with improved resistance to fracture failures . These efforts included optimizing the inorganic filler level, reducing filler size to nanoscale, adding different types of fillers such as fibers, whiskers and nanotubes, improving the polymer matrix and optimizing the polymerization reactions. These strategies have yielded composites with better mechanical performance and improved longevity. Nonetheless, composites are still brittle and prone to fracture, especially in large-sized restorations in high load-bearing locations . Therefore, there is a strong need to inhibit crack growth and fracture failures in dental resin-based restorations .
Self-healing polymers have the promise of autonomic repair to heal cracks and damage . They have the built-in capability to repair damage and recover the load-bearing capability . A promising method for providing self-healing capability is by embedding microcapsules into the composite . Each microcapsule has an external shell that encapsulates a healing liquid inside. When the polymer experiences damage and cracking, the microcapsules could be ruptured by the damage. They release the healing liquid which flows into the crack planes and the damage site, exposing itself to the catalyst embedded in the polymer matrix. Self-healing then occurs due to the polymerization of the healing agent, which fills and bonds the crack together and inhibits the crack propagation . In a pioneering study, dicyclopentadiene (DCPD) was encapsulated in a poly(urea-formaldehyde) (PUF) shell to form microcapsules . Grubb’s catalyst, a transition metal carbine complex, was used in an epoxy matrix to trigger the polymerization of the released DCPD, which achieved a good self-healing efficiency . Since then, several other studies were reported on microcapsules of various shell and healing liquid compositions, with potential applications ranging from microelectronics to aerospace , as well as bone cement where effective rebonding of the crack was achieved, with the healed specimens reaching approximately 75% of the original fracture toughness .
For dental use, efforts were made to incorporate DCPD-containing microcapsules and Grubb’s catalyst into a composite, achieving a recovery of 57% of the original fracture toughness of composite . Another study tested the mechanical properties of dental resins with DCPD-containing microcapsules, showing that the use of microcapsules did not affect the mechanical performance of resin matrix . However, literature search revealed no further report on the use of DCPD and Grubb’s catalyst in dental materials, likely due to biocompatibility concerns. Toxicity from the self-healing agent inside the microcapsules as well as the catalyst present in the composite needs to be considered . The DCPD toxicity , Grubb’s catalyst toxicity, and their high cost remain challenges in using them in dental composite. Another study developed nanocapsules with a polyurethane (PU) shell containing the triethylene glycol dimethacrylate (TEGDMA) liquid, demonstrating that the addition of nanocapsules into a dental adhesive increased the dentin bond strength . However, these nanocapsules contained no catalyst for polymerization, and hence no self-healing effect on adhesive resin was reported . To date, there has been no report on the use of polymerizable dental monomers as healing liquid with proven biocompatibility in dentistry to develop a resin demonstrating a substantial self-healing capability.
The healing liquid should have a relatively low viscosity to flow and fill the cracks in the resin matrix. TEGDMA is flowable and has been used as a dental monomer with acceptable biocompatibility. Furthermore, TEGDMA can form a polymer via free-radical initiation by using a peroxide initiator and a tertiary amine accelerator. A usual dental tertiary amine accelerator, N , N -dihydroxyethyl- p -toluidine (DHEPT), has good solubility and stability in TEGDMA. Therefore, the present study proposed to test DHEPT incorporation in TEGDMA to serve as the self-healing liquid inside microcapsules for the first time. In the present study which was our first study, we focused on the synthesis and characterization of the self-healing microspheres, incorporation into resin, physical and mechanical properties, and fibroblast cytotoxicity. Then in our second study, we further added calcium phosphate and antibacterial monomer to the self-healing resin, and tested oral biofilm response .
Accordingly, the objectives of this study were to investigate TEGDMA-DHEPT as a self-healing liquid in PUF microcapsules for dental applications and to examine the mechanical properties and self-healing efficacy. The following hypotheses were tested: (1) The new TEGDMA-DHEPT-in-PUF microcapsules could be incorporated into a dental resin without decreasing the mechanical properties of the resin; (2) The microcapsule-containing resin would have a high self-healing capability after fracture; (3) The microcapsule-containing resin would have a low cytotoxicity against human fibroblasts.
Materials and methods
Synthesis of microcapsules
Microcapsules were prepared by in situ polymerization of formaldehyde and urea, following a previous study . DHEPT (Sigma–Aldrich, St. Louis, MO) at 1% mass fraction was added to TEGDMA monomer (Esstech, Essington, PA). At room temperature, 50 mL of distilled water and 13 mL of a 2.5% aqueous solution of ethylene-maleic anhydride (EMA) copolymer (Sigma–Aldrich) were mixed in a 250 mL round bottom glass flask. The flask was suspended in a water bath on a hotplate (Isotemp, Fisher Scientific, Pittsburg, PA). The EMA solution was used as a surfactant to form an “oil-in-water” emulsion (“oil” being TEGDMA-DHEPT). Under agitation by a magnetic stir bar (diameter = 7.8 mm, length = 50 mm, Fisher Scientific) at 300 rpm, the shell-forming material urea (1.25 g), ammonium chloride (0.125 g) and resorcinol (0.125 g) (Sigma-Aldrich) were added into the solution. The resorcinol was added in the reaction of shell formation to enhance the rigidity of the shells . The pH was adjusted to 3.5 via drop-wise addition of 1 M sodium hydroxide solution. Then, the agitation rate was increased to 400 rpm, and 30 mL of the TEGDMA-DHEPT liquid was added into the flask. A stabilized emulsion of fine TEGDMA-DHEPT droplets was formed after 10 min of agitation. Then, 3.15 g of a 37% aqueous solution of formaldehyde (Sigma–Aldrich) was added, and the flask was sealed with aluminum foil to prevent evaporation. The temperature of the water bath was raised to 55 °C and the shell material was isothermally polymerized for 4 h under continuous agitation . In this process, ammonium chloride catalyzed the reaction of urea with formaldehyde to form PUF at the oil–water interface to develop the shell . The microcapsules thus obtained were rinsed with water and acetone repeatedly, vacuum-filtered, and air-dried for 24 h in a hood.
Characterization of microcapsules
An optical microscope (TE2000-S, Nikon, Japan) was used to examine the microcapsules. The microcapsule diameters were measured using the optical microscope together with an image analysis software (Nis-Elements BR2.30, Nikon, Japan). In addition, the surface morphology of TEGDMA-filled microcapsules was examined with scanning electronic microscopy (SEM, Quanta 200, FEI, Hillsboro, OR). Microcapsules were spread onto an adhesive tape and sputter-coated with gold. Some microcapsules were placed on an adhesive tape and cut open with a razor blade to facilitate the examination of the shell structure, following a previous study .
The ability of the encapsulated TEGDMA to polymerize was quantified by near-infrared (NIR) spectroscopy (Nicolet 6700, Thermo Scientific, Waltham, MA). A mixture of microcapsules with 0.5% by mass of benzoyl peroxide (BPO) initiator powder (the catalyst) (Sigma–Aldrich) were broken by manual spatulation and placed in a 6 mm × 3 mm × 2 mm Teflon mold. NIR spectra were acquired immediately after resin paste mixing and at 24 h post-cure in an FTIR spectrometer (Nicolet 6700, Thermo Scientific). The degree of vinyl conversion (DC) was calculated from the % change in the integrated peak area of methacrylate CH 2 absorption band, normalized by thickness of the specimen before and after curing, following a previous study .
The yield of microcapsules was calculated as the ratio of the dry weight of total microcapsules of each batch divided by the weight of the initial substances , i.e. , urea, resorcinol, ammonium chloride, formaldehyde, TEGDMA and DHEPT. Microcapsules loaded with TEGDMA were dispersed in acetone and sonicated at 40 Hz for 5 min (Branson, Fisher Scientific). This broke the microcapsules, and the TEGDMA and DHEPT were dissolved in the acetone solvent. After vacuum filtration and drying for 1 d, the undissolved microcapsule shell fragments were collected and weighed. The TEGDMA-DHEPT mass was calculated as the initial weight of the microcapsules minus the shell weight. The TEGDMA-DHEPT mass fraction in the microcapsules was calculated as the TEGDMA-DHEPT mass divided by the total mass of the microcapsules .
To assess the stability of the microcapsules, additional microcapsules were exposed to air at ambient temperature for 30 d. The TEGDMA-DHEPT mass fraction in the aged microcapsules was again calculated following the aforementioned procedures.
The thermal stability of the microcapsules was tested using differential scanning calorimetry (DSC, 2920MDSC, TA, New Castle, DE) to measure the heat flow. Five mg of microcapsules was placed in an air-tight aluminum pan and heated at a heating rate of 10 °C/min from 0 °C to 350 °C in a nitrogen environment. The DSC diagram was recorded by the DSC analysis instrument. With the increase in temperature, any exothermic and endothermic events were recorded on the DSC diagram, thus providing information on how thermally stable the microcapsules were, and at what temperature they became unstable. All assessments were conducted using 6 batches of samples ( n = 6).
Fabrication of self-healing dental resin
Bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) and TEGDMA monomers were mixed at a 1:1 mass ratio. The mixture was rendered light-curable by adding 1% by mass of a photo-initiator, phenyl bis(2,4,6-trimethylbenzoyl) phosphine oxide (BAPO, Sigma–Aldrich). Then, 0.5% benzoyl peroxide (BPO) was also added to the monomer mixture as the self-healing initiator. BPO could react with DHEPT from the microcapsules in the case of microcapsule rupture to cause self-healing. The 0.5% of BPO was selected because it did not decrease the mechanical properties of the resin and it was sufficient to trigger the free-radical polymerization in a preliminary test.
Microcapsules were mixed into the photo-activated BisGMA-TEGDMA monomers at the following mass fractions: 0%, 5%, 10%, 15% and 20%. Microcapsule mass fractions greater than 20% were not used because the mechanical properties of the resin were degraded in preliminary studies. For mechanical testing, each paste was placed in a stainless steel mold of 2 mm × 2 mm × 25 mm and covered with a Mylar strip. Each specimen was photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side of the mold. The specimens were demolded and stored in distilled water at 37 °C for 24 h prior to testing.
A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used to fracture the specimens in three-point flexure using a span of 10 mm and a crosshead speed of 1 mm/min. Flexural strength S was measured as: S = 3 P max L /(2 bh 2 ), where P max is the load-at-failure, L is span, b is specimen width and h is thickness. Elastic modulus E was measured as: E = ( P / d )( L 3 /[4 bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region of the load-displacement curve. The specimens were taken out of the water and fracture while being wet. Six specimens of each group were tested .
Fracture toughness testing
Fracture toughness K IC was measured via a single edge V-notched beam (SEVNB) method under mode I condition . A notch with a depth of approximately 500 μm was machined into a resin bar of 2 mm × 2 mm × 25 mm using a diamond blade with a 150 μm thickness (Buehler, Lake Bluff, IL). Then, a 3-μm diamond paste (Buehler) was placed into the notch tip, and a new sharp razor blade was used to cut the notch further to a total depth of about 700–800 μm. According to previous studies using the same method, this produced a relatively sharp notch with a tip radius of approximately 20 μm . For each specimen, the notch length was measured using an optical microscope (TE2000-S, Nikon) on both sides of the specimen and then the notch length was averaged. The notched specimens were tested in the same three-point fixture with the notch on the tensile side, and the loading pin aligned with the notch under a 3× magnifier. K IC was calculated following an established SEVNB method . The photo-cured BisGMA-TEGDMA resins with 0–20% of microcapsules were tested. This yielded the original virgin K IC of the specimens, K IC-virgin .
In order to test the self-healing efficacy, immediately following specimen fracture, the two halves of the specimen were placed back into the same mold which was labeled and was originally used to make the same specimen. This is similar to the case of a resin-based restoration in a tooth cavity, where the cracked restoration would stay in the tooth cavity to allow the released healing liquid to heal the crack. Because the fracture ruptured the microcapsules, the released TEGDMA-DHEPT from the microcapsules would react with the BPO in the resin matrix. This would cause the polymerization of the released liquid to heal and bond the two cracked planes into one cohesive specimen. This healed sample was placed in a humidor at 37 °C for 24 h. The notch length was again measured and made sure that it was the same as the virgin notch length of the same specimen. Then the healed bar was tested again using the same flexural method to measure the K IC . This yielded the healed value, K IC-healed .
The self-healing efficiency ( η ) was assessed following a previous study as :
η = K IC-healed K IC-virgin
The fractured surfaces of selected self-healed specimens were examined via SEM after sputter-coating with gold. In addition, the fractured and unhealed surfaces, which were rinsed with ethyl alcohol immediately after fracture to remove the released healing agent, were also examined for investigation of fracture mechanisms.
The cytotoxicity of the five groups of resins containing 0%, 5%, 10%, 15% and 20% of microcapsules was examined by measuring cell survival and proliferation in vitro via a MTT assay . To prepare resin specimens for cytotoxicity testing, the cover of a sterile 96-well plate was used as molds, following a previous study . Fifty μL of resin paste of each group was placed into the bottom of the dent and covered with a Mylar strip, then light-cured for 30 s (Optilux VCL 401, Demetron Kerr, Danbury, CT). This formed a disk of about 8 mm in diameter and 0.5 mm in thickness. The disks were sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). Then, six disks of each group were immersed in 10 mL of a commercial fibroblast culture medium (ScienCell, San Diego, CA), referred to as FM. The medium was agitated for 24 h at 37 °C to obtain resin eluents, following a previous study . The original extract solution was then serially diluted for the cytotoxicity test . Three solutions were prepared at dilutions of 32-fold (1 part of original extract + 31 parts of fresh FM), 64-fold, and 128-fold, respectively, following a previous study . The reason for choosing 128-fold dilution was that the amount of saliva flow for an average person is approximately 1000–1500 mL per day . The 128-fold dilution yielded to a total solution volume of 1280 mL for the six resin disks. The 32-fold and 64-fold corresponded to a total solution of 320 mL and 640 mL respectively, which would be equivalent to 1/4 and 1/2 of the normal saliva amount per day, to take into account of reduced saliva production for some patients. The total resin volume of the six disks was 151 mm 3 . Hence, the resin volume divided by the total solution volume for the three solutions was 0.12, 0.24, and 0.47 mm 3 /mL, respectively. Fresh FM without any resin eluent (resin/solution volume ratio = 0 mm 3 /mL) served as the control in cell culture.
Human gingival fibroblasts (HGF, ScienCell) were cultured in a growth medium consisting of FM supplemented with 2% fetal bovine serum, 100 IU/mL penicillin and 100 IU/mL streptomycin. They were seeded into the wells of 96-well micro-plates at a density of 4000 cells/well . After 24 h of incubation at 37 °C in a humid atmosphere with 5% CO 2 in air, the culture medium in the 96-well microplates was removed and replaced with 100 μL of the aforementioned resin extract solutions. Then the cells were cultured for another 48 h. To each well was added 20 μL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Sigma-Aldrich) solution at a concentration of 5 mg/mL . After incubating in a dark room for 4 h, the unreacted dye was replaced by adding150 μL/well of dimethylsulfoxide (DMSO, Sigma-Aldrich). The plate was gently shaken until the crystals were completely dissolved. Then the absorbance of the solutions was measured spectrophotometrically using a microplate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA) at 492 nm. Pure FM without any resin eluent was used as control to culture fibroblasts, and its absorbance was set as 100%. Cell viability was assessed as A resin / A control , where A resin was the absorbance of cells cultured with resin eluents, and A control was the absorbance of cells cultured in FM without resin eluents .
One-way analysis of variance (ANOVA) was performed to detect the significant effects of the variables. Tukey’s multiple comparison test was used at a p value of 0.05.