Small-sized fibroblast growth factor-2 (FGF-2)-loaded particles used as carriers.
FGF-2-loaded particles were added in the 4-META/MMA-TBB resin.
Sustainable FGF-2 release from the experimental resin was observed.
The experimental resin promoted osteoblast proliferation.
Addition of the particles showed no influence on physical properties of the resin.
Non-biodegradable particles comprising hydroxyethyl methacrylate (HEMA) and trimethylolpropane trimethacrylate (TMPT) have been reported as useful carriers for fibroblast growth factor-2 (FGF-2). They have also been successfully incorporated into the 4-[2-(methacryloyloxy)ethoxycarbonyl]phthalic anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin to promote tissue regeneration. However, smaller particles are required to obtain restorative materials acceptable for clinical use. The aim of this study was to investigate the ability of the 4-META/MMA-TBB resin that comprises small FGF-2-loaded particles to release FGF-2 and promote cell proliferation. In addition, the bonding and physical properties of the experimental resin were evaluated.
The small particles loaded with FGF-2 were newly fabricated and incorporated into the commercial 4-META/MMA-TBB resin. Release profiles of FGF-2 from the experimental resins were assessed, and the cell proliferation cultured with the eluate was evaluated. The bonding and physical properties of the resins were evaluated using shear bond strength and three-point bending tests, and by measuring the curing time, water absorption, and water dissolution.
Sustained release of FGF-2 from the experimental resins for two weeks was observed, and the released FGF-2 was demonstrated to promote cell proliferation. All bonding and physical properties of the 4-META/MMA-TBB resins were found acceptable for clinical use.
The small FGF-2-loaded particles incorporated into the 4-META/MMA-TBB resin had the same abilities to release FGF-2 and proliferate cells, as those exhibited by the conventionally sized particles. In addition, there were no adverse influences on bonding and physical properties, suggesting that the bioactive adhesive resin was acceptable for clinical use.
Dental adhesive is used not only for restorative and prosthesis treatments but also for the adhesion of fractured roots [ ] or root end fillings [ ], which are used in contact with periodontal tissues. Although the bonding property of adhesive resins has improved, they still cannot facilitate the healing of surrounding tissues. To increase the success rate of treatments utilizing these adhesive resin types to reconstruct fractured roots or root end fillings, the ability to promote tissue regeneration would be advantageous. In our previous study, we fabricated poly(2-hydroxyethyl methacrylate (HEMA)/trimethylolpropane trimethacrylate (TMPT)) particles as the non-biodegradable carrier for antimicrobials and growth factors [ ]. We demonstrated the ability of fibroblast growth factor-2 (FGF-2)-loaded polyHEMA/TMPT particles to continuously release FGF-2 and induce tissue regeneration when the particles were combined with a commercial 4-[2-(methacryloyloxy)ethoxycarbonyl]phthalic anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin [ ].
The 4-META/MMA-TBB resin exhibited high biocompatibility with periodontal tissues and excellent bonding/sealing capabilities in wet conditions [ , ]. Among a plethora of adhesive resins, the 4-META/MMA-TBB resin was suitable as a base resin that can incorporate FGF-2-loaded polyHEMA/TMPT particles. However, because the average diameter of the polyHEMA/TMPT particles that we previously fabricated was approximately 600 μm, these particles were excessively large to maintain the bonding ability and physical properties of the 4-META/MMA-TBB resin. To prevent deterioration of the basic resin properties, smaller polyHEMA/TMPT particles were required to achieve adhesive resins acceptable for clinical use. The purpose of this study was to fabricate small FGF-2-loaded polyHEMA/TMPT particles and evaluate the ability the of the 4-META/MMA-TBB resin to release FGF-2 and promote the proliferation of osteoblast-like cells. Furthermore, the influences of the small polyHEMA/TMPT particles on bonding abilities and physical properties were confirmed.
Materials and methods
Preparation of polyHEMA/TMPT particles
The polyHEMA/TMPT particles were prepared by following the steps indicated in the study conducted by Takeda et al. [ ]. HEMA (Tokyo Chemical Industry Inc., Tokyo, Japan) and TMPT (Shin-Nakamura Chemical Inc., Wakayama, Japan) were mixed at a weight ratio of 90/10. The mixture of monomers was polymerized at 120 °C for 2 h; subsequently, post-polymerization was observed to occur for 16 h at -0.1 MPa. The obtained cured product was roughly pulverized with a hammer-type grinder (MF 10 basic S1, IKA, Staufen, Germany). Subsequently, the particles were finely pulverized using a planetary rotating ball mill (LP-1, Ito Seisakusyo Inc., Tokyo, Japan), and particles that passed through a 500-mesh sieve were collected. The fabricated particles were observed using a scanning electron microscope (SEM) (JSM-6390, JEOL, Tokyo, Japan) at 5 kV under×1000 magnifications. The average diameter and size distribution of the particles were measured by laser diffraction particle size analyzer (LS 13 320, Beckman Coulter Inc., CA, USA).
Assessment of FGF-2 release from polyHEMA/TMPT particles
To load FGF-2, 30 mg of polyHEMA/TMPT particles were immersed in a 500 μg/mL FGF-2 solution (Fiblast, Kaken Pharmaceutical Inc., Tokyo, Japan), and stored at 4 °C for 24 h before being washed with distilled water. After storage at −80 °C for 24 h, the particles were dried in a vacuum dryer (JFD-310, JEOL Inc., Tokyo, Japan) at -4 °C for 12 h.
Thirty mg of polyHEMA/TMPT particles loaded with FGF-2 were immersed in 200 μL of distilled water and incubated at 37 °C to assess the release profile of FGF-2. The distilled water was replaced at different intervals, namely: 15 and 30 min, 1, 3, 6, and 12 h, and 1 and 2 d; subsequently, the concentrations of FGF-2 in the eluates were measured using the Micro BCA protein assay kit (Thermo Fisher Scientific Inc., Kanagawa, Japan). These experiments were carried out five times.
Preparation of the experimental resin incorporating FGF-2-loaded polyHEMA/TMPT particles
The experimental resin was prepared by adding FGF-2-loaded polyHEMA/TMPT particles to a commercial 4-META/MMA-TBB resin powder (Super-Bond, Sun Medical Inc., Shiga, Japan) at 10 or 30 (wt)%. The polyHEMA/TMPT particles and resin powder were mixed in a monomer solution with the original polymer / monomer ratio of the Super-Bond to catalyst. The mixture was placed into silicon-based molds (diameter: 5.0 mm; height: 0.5 mm) and resin discs were prepared by curing the Super-Bond inside the mold. The cured discs were briefly immersed into 200 μL of distilled water at 37 °C, and the eluates were collected at each time point until day 14 and replaced with fresh distilled water. The concentrations of FGF-2 in the eluates were measured. These experiments were performed a total of five times.
Effects of FGF-2 on the proliferation of osteoblast-like cells
The MC3T3-E1 cell (Osteoblast-like cell line) was cultured in an α-minimum essential medium (α-MEM) with L-glutamine and phenol red (Wako Pure Chemical Industries Inc., Osaka, Japan). The adhesive resin discs incorporating 10 or 30 (wt)% of polyHEMA/TMPT particles were immersed into 100 μL of the medium at 37 °C for 15 min, and then collected to evaluate the effects of the released FGF-2 on cells. MC3T3-E1 cells were seeded in 96-well plates at 2 × 10 4 cells/well and cultured for 24 h. Next, the entire medium was removed and replaced with a fresh 180 μL α-MEM containing 20 μL eluate and cultured for another 24 h. The medium was collected after immersing the polyHEMA/TMPT particles that were not loaded with FGF-2 for 15 min. Further, the cells cultured with the medium only were used as controls. The cell proliferation was evaluated using MTT assays. Further, 20 μL of 3-(4,5-dimethylthiazoly1-2)-2,5-diphenyl-tetrazolium bromide solution (Sigma-Aldrich Inc., St. Louis, MO, USA) was briefly added to each well, and the plates were incubated for 4 h at 37 °C. After suctioning the medium, 200 μL of dimethyl sulfoxide (Wako Pure Chemical Industries Inc., Osaka, Japan) was added to each well. The absorbance at 560 nm was then measured. These experiments were also carried out five times.
Influences of polyHEMA/TMPT particles on bonding ability
To assess the incorporation of polyHEMA/TMPT particles on the dentin-bonding ability of the Super-Bond, the experimental resin incorporated 10 or 30 (wt)% of the particles. The crowns of bovine incisors were embedded in the chemically-cured acrylic resin and the dentin surface was exposed through polishing with an 80-grit waterproof abrasive paper. The flat surface of the tooth was prepared using a 600-grit waterproof abrasive paper and cleaned with ultrasound. After the treatment with 10% citric acid and 3% ferric chloride solution (Green Activater, Sun Medical Inc.,) was achieved by following the product instructions (treated for 10 s, then washed and air dried), stainless steel rods (3 mm in diameter) were bonded to the dentin surface with the adhesive resin under the load of 10 N for 10 s. The bonded specimens were kept in water at 37 °C for 24 h or 7d, and a shear bond strength test was performed using a tabletop testing machine (EZ Test; Shimadzu) at a crosshead speed of 1 mm/min.
The shear bond strength was calculated by dividing the load by the bonded area (7.07 mm 2 ). The Super-Bond without the particle represented the control. These experiments were carried out ten times.
Influences of polyHEMA/TMPT particles on mechanical properties
The ultimate flexural strength was evaluated by using a three-point bending test in a universal testing machine (INSTRON-5544, Instron Pty Ltd., Bayswater, Melbourne, Australia). The flexural test was conducted by following the ISO 4049:2009 standard. The cured specimen was prepared using a metal mold (25 × 2 × 2 mm) and stored in water at 37 °C for 24 h or 7 d. The Super-Bond without the particle represented the control. The loading force was applied to the specimens at a crosshead speed of 1 mm/min. These experiments were also performed five times.
Curing time measurement
The temperature of the experimental resin during curing was measured using differential scanning calorimetry (DSC). The experimental resin was mixed and put on the steel dish and then the dish was placed in the DSC apparatus (DSC-60, Shimazu Inc., Kyoto, Japan) for 30 s. The time to reach the peak temperature was considered as the curing time. These experiments were carried out five times.
Water absorption and dissolution
Water uptake by the experimental resins was measured according to the ISO 4049: 2009 standard. The cured specimen (15 mm × 15 mm × 1.0 mm) was carefully polished with fine sandpapers and then stored in a desiccator at 37 °C. The sample weights were measured after being conditioned in a desiccator at 37 °C for 22 h and at 23 °C for 2 h with an electric balance; the resulting accuracy was 0.01 mg. Subsequently, the specimen was immersed in a water bath at 37 °C for 7 d. The samples were then removed from the bath, dried with filter papers, and weighed. The water uptake was calculated and the specimen was dried again until a constant weight was obtained, after which the dissolution amount was calculated. These experiments were also carried out five times.
Statistical analyses were performed using SPSS Statistics 21 (IBM, Chicago, IL, USA). Cell proliferation, curing time test, and water sorption/ dissolution results were evaluated using analysis of variance (ANOVA) and the Tukey’s honesty significant difference (HSD) test. The shear bond strength and three-point bending tests results were analyzed using the Kruskal-Wallis H test. P -values ≥ 0.05 were statistically insignificant.
Morphology and size of fabricated polyHEMA/TMPT particles
SEM images of the fabricated polyHEMA/TMPT particles are shown in Fig. 1 A. The particles had irregular shapes and their average diameter was 8.7 ± 6.5 μm ( Fig. 1 B). The majority of the Super-Bond powders were spherical and contained some irregular shaped particles ( Fig. 1 C) and the ratio of diameter 8−9 μm was high ( Fig. 1 D).