Surface pre-reacted glass-ionomer (S-PRG)-containing dental materials, including composite and coating resins have been used for the restoration and/or prevention of dental cavities. S-PRG is known to have the ability to release aluminum, boron, fluorine, silicon, and strontium ions. Aluminum ions are known to be inhibitors whereas boron, fluorine, silicon, and strontium ions are known to be promoters of mineralization, via osteoblasts. However, it remains to be clarified how an aqueous eluate obtained from S-PRG containing these ions affects the ability of mesenchymal stem cells (MSCs), which are known to be present in dental pulp and bone marrow, to differentiate into osteogenic cell types. The present study demonstrated that 200- to 1,000-fold-diluted aqueous eluates obtained from S-PRG significantly upregulated the mRNA expression level of the osteogenic differentiation marker alkaline phosphatase in human MSCs (hMSCs) without exhibiting the cytotoxic effect. In addition, the 500- to 1,000-fold-diluted aqueous eluates obtained from S-PRG significantly and clearly promoted mineralization of the extracellular matrix of hMSCs. It was additionally demonstrated that hMSCs cultured on the cured resin composites containing S-PRG fillers exhibited osteogenic differentiation in direct correlation with the weight percent of S-PRG fillers. These results strongly suggested that aqueous eluates of S-PRG fillers promoted hard tissue formation by hMSCs, implicating that resins containing S-PRG may act as a useful biomaterial to cover accidental exposure of dental pulp.
Dental restoration is frequently performed using resin composites to repair missing parts of teeth caused by dental trauma or traumatic damage. Resin composites consist of three different materials: An organic phase (matrix), a dispersed phase (inorganic filler), and an interfacial phase (coupling agent to bind the filler to the organic resin) (
Surface pre-reacted glass-ionomer (S-PRG) fillers are prepared by an acid-based reaction between fluoro-boro-aluminosilicate glass and polyacrylic acid solution (
Here, we examined how aqueous eluates obtained from S-PRG affect the viability and osteoblastic differentiation of MSCs. In addition, we evaluated the status of osteogenic differentiation of human MSCs (hMSCs) cultured on S-PRG-containing composite resins.
Human bone marrow-derived MSCs, UE7T-13 cells immortalized by overexpression of human papillomavirus E7, and human telomerase reverse transcriptase (hTERT) (
A total of 20 g of powdered S-PRG fillers was thoroughly mixed with 20 ml of DMEM in a 50-ml plastic centrifuge tube using a tube rotator (rgos. Technologies, Inc., Elgin, IL, USA) at 40 rpm for 24 h at room temperature. Then, the suspension of the S-PRG fillers in DMEM was aliquoted into 2-ml centrifuge tubes, and the filler particles in the suspension were centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was sterilized by filtration using a nylon filter membrane with a pore size of 0.22 µm (33 mm diameter; Sigma-Aldrich).
The cell viability was evaluated using an alamarBlue assay (AbDSerotec, Oxon, UK) according to the manufacturer's instruction. The assay includes an indicator that fluoresces and undergoes colorimetric change when reduced by mitochondrial respiration, which is proportional to the number of living cells. The cells were cultured with DMEM containing 10% alamarBlue solution to evaluate the viability of the cells, and the cells were cultured for an additional 4.5 h. The absorbance in each well was measured using a micro plate reader (Tosoh Corp., Tokyo, Japan). The data are shown as values of Abs570-Abs600. Each experiment was repeated three times with six wells dedicated to each time point.
The total RNA from UE7T-13 cells was isolated with ISOGENII reagent (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions. First-strand cDNA was synthesized from total RNA using the PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan). PCR was subsequently performed on a Thermal Cycler Dice Real Time System (Takara Bio, Inc.) using SYBR Premix Ex Taq II (Takara-Bio) with specific oligonucleotide primers (human ALP, 5′-GGACCATTCCCACGTCTTCAC-3′ (forward) and 5′-CCTTGTAGCCAGGCCCATTG-3′ (reverse); human GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-ATGGTGGTGAAGACGCCAGT-3′ (reverse)). The mRNA expression of ALP was normalized to that of GAPDH, and the relative expression levels are shown as fold increase or decrease relative to the control.
ECM mineralization was evaluated by Alizarin red S (A5533; Sigma-Aldrich) staining after the fixation with 4% paraformaldehyde. Then, the dye bound to the calcified nodules was extracted by 5% formic acid. The absorbance of the extracted solution was measured using a micro plate reader. The data are shown as values of Abs405-Abs600. Each experiment was repeated three times with five wells dedicated to each time point.
Elemental analysis of ions (Al, B, F, Na, Si, and Sr, which were presumed to be contained in the glass core composition) released from the S-PRG filler was performed using an inductively coupled plasma (ICP) atomic emission spectrometer (ICP-AES: ICPS-8000; Shimadzu Corporation, Kyoto, Japan) or fluoride electrode method (fluoride electron, model 9609BN; pH/ion meter, model 720A, Orion Research).
Analysis using ICP was conducted after preparing calibration curves corresponding to each element (standard solution concentrations; Si: 0, 0.5, 1, 5 ppm; Sr: 0, 5, 20, 50 ppm; B: 0, 10, 50, 100 ppm; Al: 0, 0.5, 5, 10 ppm; Na: 0, 0.5, 20, 50 ppm). Fluoride was analyzed using a fluoride ion electrode method after preparing calibration curves (standard solution concentrations: 0.1, 1, 5, 10 ppm). An ionic strength adjuster (TISAB III, Orion Research) was added in the proportion of 0.1 ml of ionic strength adjuster to 1 ml of test liquid.
Resin composite discs with a diameter of 20 mm and a thickness of 2 mm containing S-PRG fillers at 0, 10, 30, 50, and 70% by weight were casted into a mold made from polytetrafluoroethylene and cured. Then, the surface of the cured composite resin discs was polished with waterproof sandpaper no. 400. The cured resin composite discs were disinfected by immersion of the discs in 99.5% ethanol for 24 h. The disinfected resin discs were inserted into each well of 12-well plastic cell culture plates.
The data are presented as mean ± standard deviation (n=3, 6, or 8). The data were statistically analyzed by Dunnett's multiple comparison test, and P<0.05 was considered to indicate a statistically significant difference. The results shown in all experiments are representative of at least three separate experiments.
The alamarBlue assay revealed that the non-, 2-, 5-, and 10-fold-diluted DMEM eluate decreased the viability of UE7T13 cells to 17.3, 62.2, 80.6, and 74.2% of the control, respectively, 48 h after its administration (
UE7T-13 cells were cultured with non-diluted DMEM or the 100- to 1,000-fold-diluted DMEM eluate of S-PRG fillers supplemented with 10% FBS as described in the Materials and Methods. The 200-, 500- and 1,000-fold-diluted DMEM eluate significantly increased the expression of ALP mRNA to 157.3, 193.3, and 194.5% of the control, respectively, 72 h after administration in a dilution-dependent manner (
Alizarin red staining revealed that the 500- and 1,000-fold-diluted DMEM eluates promoted calcium deposition in the ECM of UE7T-13 cells (
As shown in
In order to examine whether resin composites containing S-PRG fillers retain an advantage in inducing osteogenic differentiation of MSCs, we evaluated the mRNA expression status of ALP in UE7T-13 cells under co-incubation with cured resin composites containing various volumes of S-PRG fillers as indicated (
Hakki
Zou
Bao
Nakade
Sun
We also found that ALP mRNA expression in the hMSCs cultured in the diluted DMEM eluate of S-PRG filler decreased at the 2,000-, to 8,000-fold-dilution range of the S-PRG eluate in comparison with ALP mRNA expression in the hMSCs cultured in the 1,000-fold diluted eluate in a dilution rate-dependent manner (data not shown). On the other hand, hMSCs cultured on the cured resin composites containing S-PRG fillers exhibited osteogenic differentiation in direct correlation with the weight percent of S-PRG fillers (
Calcium hydroxide is often used for endodontic treatments involving dental pulp capping, and known to induce calcification of extracellular matrix between dental pulp cells involving MSC-like cells (
The present study was supported in part by JSPS KAKENHI grant nos. 26670852 and 16H05534 to A.I., nos. 25463053 and 16K11654 to N.C., no. 26462823 to S.K., no. 15K20606 to H.K., no. 22592076 to M.K., and a Grant-in-Aid for Strategic Medical Science Research Centre from the Ministry of Education, Culture, Sports, Science and Technology of Japan, 2010–2014.
surface pre-reacted glass-ionomer
mesenchymal stem cells
human mesenchymal stem cells
poly-(lactide-co-glycolide-acid)
extracellular matrix
alkaline phosphatase
transforming growth factor
human telomerase reverse transcriptase
Dulbecco's modified Eagle's medium
fetal bovine serum
inductively coupled plasma
osteoblasts
Eluate from S-PRG fillers showed cytotoxicity in UE7T-13 cells in direct correlation with the volume percent of the DMEM eluate. An alamarBlue assay was used to examine the viability of UE7T-13 cells. The cells were seeded into 96-well cell culture plates at a density of 1×104 cells per well and cultured for 24 h in medium supplemented with 10% FBS. The medium was replaced with DMEM containing eluate from S-PRG fillers, diluted with normal DMEM at the ratios of 1 to 1/800. Then, the diluted eluate of S-PRG fillers was supplemented with 10% FBS, and the cells were subsequently cultured for 48 h. Finally, the medium was replaced with DMEM containing 10% alamarBlue solution to evaluate the viability of the cells as described in the Materials and methods. Data represent the mean ± standard deviation (
Eluate from S-PRG fillers induced mRNA expression of osteoblastic markers in UE7T-13 cells. An aqueous eluate of S-PRG fillers was prepared as described in the Materials and Methods. UE7T-13 cells were seeded into 12-well cell culture plates at a density of 1×105 cells per well and cultured for 24 h in medium supplemented with 10% FBS. The medium was replaced with DMEM containing eluate from S-PRG fillers, diluted with normal DMEM at the ratios of 1/100 to 1/1,000. Then, the diluted eluate in each well of the cell culture plates was supplemented with 10% FBS, and the cells were subsequently cultured for 72 h. Then, the total RNA from the cells was isolated, and the relative mRNA expression of the osteoblastic marker ALP was evaluated using qRT-PCR as described in the Materials and methods. The mRNA expression of ALP was normalized to that of GAPDH. Data represent the mean ± standard deviation (
Eluate from S-PRG fillers promoted mineralization of ECM by UE7T-13 cells. UE7T-13 cells were seeded into 48-well cell culture plates at a density of 1×105 cells per well and cultured for 24 h in medium supplemented with 10% FBS. The medium was replaced with DMEM containing S-PRG fillers, non-diluted or diluted with normal DMEM to reach the indicated ratios. Then, the non-diluted or diluted eluate of S-PRG fillers in each well of 12-well cell culture plates was supplemented with 10% FBS, and the cells were subsequently cultured for 14 days. (A) Bone matrix mineralization was evaluated by Alizarin red S staining as described in the Materials and methods. Scale bar: 100 µm. (B) The dye bound to the calcified nodules was extracted by 5% formic acid. The absorbance of the extracted solution was measured as described in the Materials and methods. Data represent the mean ± standard deviation (
Resin composites containing S-PRG fillers significantly promoted ALP expression in UE7T-13 cells in direct correlation with the weight percent of S-PRG fillers. The cured resin composite discs were made as described in the Materials and methods and inserted into each well of 12-well plastic cell culture plates. UE7T-13 cells were seeded on the disinfected resin discs with DMEM supplemented with 10% FBS and antibiotics at a density of 1×105 cells per well and further cultured for 48 h. Then, the total RNA from the cells was isolated, and the relative mRNA expression of the osteoblastic marker ALP was evaluated using qRT-PCR as described in the Materials and methods. The mRNA expression of ALP was normalized to that of GAPDH. Data represent the mean ± standard deviation (
Comparison between concentrations of various ions in the non-diluted DMEM eluate of S-PRG fillers and those in normal DMEM.
Ions | Al3+ | B3+ | Na+ | Si2+ | Ca2+ | K+ | Sr2+ | F− |
---|---|---|---|---|---|---|---|---|
Normal DMEM | 0.1±0 | 0±0 | 2,640.3±200.0 | 0±0 | 75.6 0±0.8 | 206.0±6.4 | 0±0 | 0±0 |
DMEM eluate | 12.6±0.3 |
1,309.7±165.4 |
2,968.7±46.3 | 7.2±0.2 |
1.8±0.2 |
32.8±7.4 |
790.9±135.7 |
71.2±0.8 |
All values are given in ppm. Data are presented as the mean ± standard deviation (n=3).
P<0.05 were considered statistically significant. DMEM, Dulbecco's modified Eagle's medium; S-PRG, surface pre-reacted glass-ionomer