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Introduction

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have the capacity for self-renewal and differentiation into osteocytes, adipocytes and other cells (1). MSCs have been used in preclinical models for tissue engineering of bone, cartilage, muscle, marrow stroma, tendon, fat and other connective tissues (2–4). In addition, MSCs secrete a broad spectrum of bioactive macromolecules that are immunomodulatory and serve to structure regenerative microenvironments in fields of tissue injury (5). Human mesenchymal stem cells have previously been isolated and characterized from the gingiva, and gingiva-derived stem cells have been applied for tissue engineering purposes (6). Moreover, stem cells may be obtained intraorally, and gingiva is a readily accessible tissue source with a relatively high quantity of obtainable tissue (7).

Tacrolimus is a 23-member macrolide lactone (molecular weight, 803.5 Da) with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following liver, heart, kidney and small bowel transplantation (8). Tacrolimus has a narrow therapeutic window (9) and it is important to choose the right dose (10). Clinically, the doses of tacrolimus administered following kidney, heart and liver transplantation are 0.15–0.3, 0.05–0.075 and 0.10–0.2 mg/kg/day, respectively (11–14). In addition, whole blood tacrolimus concentrations were observed to be 10–15 ng/ml during months 1–3 and 5–12 ng/ml during months 4–12 with the initial tacrolimus dose of 0.15–0.2 mg/kg/day (14,15).

Tacrolimus has been shown to exert a variety of actions on bone metabolism (16). It was reported that tacrolimus causes bone loss when administered systemically (17,18). However, other studies have shown that tacrolimus promotes osteogenic differentiation (19,20). Conversely, another study demonstrated that tacrolimus was not associated with osteogenic differentiation of human heart-derived MSCs (21).

Limited information is currently available regarding the effects of tacrolimus on dental tissue, and none is available on its effects on human mesenchymal stem cells derived from the gingiva. The aim of the present study was to evaluate the effects of a broad range of concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva.

Materials and methods

Isolation and culture of stem cells derived from the gingiva

Healthy gingival tissue samples were collected from healthy patients (mean age, 51.8±18.1 years; 2 male and 2 female) and this study was reviewed and approved by the Institutional Review Board of Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea (Seoul, Korea; approval no. KC11SISI0348); informed consent was obtained from all patients. The resected gingival tissues were immediately placed in sterile phosphate-buffered saline (PBS; Welgene Inc., Daegu, Korea) with 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C. The gingival tissue was de-epithelialized, minced into 1–2 mm2 fragments, and digested in 0.2 µm filtered α-Minumum Essential Medium (MEM; HyClone; GE Healthcare Life Sciences, Chalfont, UK) with collagenase IV (Sigma-Aldrich). The cells were incubated at 37°C in a humidified incubator with 5% CO2 and 95% O2. After 24 h, the non-adherent cells were washed with PBS, and adherent cell were administered fresh medium and replaced every 2–3 days. A previous report demonstrated that these cells showed colony-forming abilities, plastic adherence, and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency (22). The cells expressed CD44, CD73, CD90, and CD105, but did not express CD14, CD45, CD34, and CD19 in flow cytometry (22).

Evaluation of cellular morphology

The cells were plated at a density of 2.0×103 cells/well in 96-well plates. The cells were incubated in α-MEM containing 15% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin in the presence of tacrolimus at final concentrations ranging of 0 (control), 0.001, 0.01, 0.1, 1, 10 and 100 µg/ml. Tacrolimus was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and filter-sterilized. Equal quantities of DMSO were added to each culture sample to offset the influence of this dissolving vehicle. The morphology of the cells was viewed under an inverted microscope (Leica DM IRM, Leica Microsystems, Wetzlar, Germany) on days 1, 3, 5 and 7. The images were saved as JPEGs.

Determination of cell viability

The cell viability analysis was performed on days 1, 3, 5 and 7. 2-(2-methoxy-4-nitrophe nyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt (WST-8 from Cell Counting kit-8; Dojindo, Tokyo, Japan) was added to the culture and the cells were incubated for 3 h at 37°C. Viable cells were identified using the CCK-8 assay, which relies on the ability of mitochondrial dehydrogenases to oxidize WST-8 to a formazan product. The spectrophotometric absorbance at 450 nm was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The tests were performed in triplicate.

Alizarin Red S staining

Cell cultures obtained on days 5 and 7 were washed twice with PBS, fixed with 70% ethanol and rinsed twice with deionized water. Cultures were stained with Alizarin Red S (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and evaluated with a microscope (Leica DM IRB; Leica Microsystems). To remove non-specifically bound stain, cultures were washed three times with deionized water and once with PBS for 15 min at ambient temperature. Bound dye was solubilized in 10 mM sodium phosphate containing 10% cetylpyridinium chloride (Sigma-Aldrich) and quantified spectrophotometrically at 560 nm (PowerWave XS2; BioTek Instruments, Inc., Winooski, VT, USA).

Statistical analysis

The data are presented as the mean ± standard deviation of the experiments. A one-way analysis of variance with post-hoc test was performed to determine the differences between the groups using a commercially available program (SPSS 12 for Windows, SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Evaluation of cell morphology

The control group showed normal fibroblast morphology on day 1 (Fig. 1). The shapes of the cells treated with 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. However, the 100 µg/ml group was markedly different when compared with the control group. The shapes of the cells in the 100 µg/ml group were rounder, and fewer cells were present. The morphology of the cells on day 3 is shown in Fig. 2. An increased number of cells were observed in each group and the shapes of the cells in the tested groups were similar to those in the control group. The results for cells on days 5 and 7 are shown in Figs. 3 and 4, respectively. Noticeable differences were observed in the 100 µg/ml group.

Figure 1 Evaluation of cell morphology on day 1 using inverted microscopy following treatment with tacrolimus of different concentrations. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group and (G) 100 µg/ml group.

Figure 2 Cell morphology on day 3 using inverted microscopy following treatment with tacrolimus of different concentrations. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group and (G) 100 µg/ml group.

Figure 3 Cell morphology on day 5 using inverted microscopy following treatment with tacrolimus of different concentrations. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group, (G) 100 µg/ml group.

Figure 4 Cell morphology on day 7 using inverted microscopy following treatment with tacrolimus of different concentrations. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group and (G) 100 µg/ml group.

Cellular viability

The CCK-8 results demonstrated cellular viability on days 1, 3, 5 and 7 are shown in Fig. 5. All groups except the 10 and 100 µg/ml group showed relatively increased cell proliferation over time. The cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml did not show any statistically significant differences compared with the control at days 1, 3 and 5 using the CCK-8 assays (P>0.05). However, growth in the presence of tacrolimus at a concentration of 100 µg/ml resulted in decreases in the CCK-8 values at days 3, 5 and 7 (P<0.05).

Figure 5 Cellular viability using cell counting kit-8 assay on days 1, 3, 5 and 7 following treatment with different concentrations of tacrolimus. *P<0.05 compared with the control group at day 3. **P<0.05, compared with the control group at day 5. #P<0.05 compared with the control group at day 7.

Mineralization assay

Mineralized extracellular deposits were minimally observed after Alizarin Red S staining on day 5 (Fig. 6). Increased mineralized deposits were noted on day 7 (Fig. 7). The quantitative results regarding bound dye on days 5 and 7 are shown in Fig. 8. The cultures grown in the presence of 0.001, 0.01, 0.1 and 1 µg/ml tacrolimus exhibited increased mineralized deposits compared with the control on day 5. The relative values of the mineralization at 0.001, 0.01, 0.1 and 1 µg/ml of tacrolimus were 110.9±10.7%, 102.1±8.4%, 106.5±8.0%, and 111.8±6.7%, respectively when the result of the control group on day 5 was considered to be 100% (100.0±9.1%). A significant decrease in mineralization was observed in the 100 µg/ml group on day 5 in comparison with the control group (P<0.05). The results for day 7 showed that treatment with tacrolimus at concentrations 0.001–1 µg/ml led to increase mineralized deposits compared with the control group. The relative value of the mineralization at 0.001, 0.01, 0.1 and 1 µg/ml of tacrolimus were 118.0±11.2, 123.6±12.3, 118.3±4.5 and 104.4±5.9%, respectively, when the result of the control group on day 7 was considered 100% (100.0±5.4%). A statistically significant decrease was observed in the 100 µg/ml group on day 7 in comparison with the control group (P<0.05).

Figure 6 Results of Alizarin Red S staining on day 5 following treatment with different concentrations of tacrolimus. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group, (G) 100 µg/ml group.

Figure 7 Results of Alizarin Red S staining on day 7 following treatment with different concentrations of tacrolimus. (A) Control, (B) 0.001 µg/ml group, (C) 0.01 µg/ml group, (D) 0.1 µg/ml group, (E) 1 µg/ml group, (F) 10 µg/ml group, (G) 100 µg/ml group.

Figure 8 Quantitative results of mineralization assay on days 5 and 7. *P<0.05 compared with the control group at day 5. **P<0.05 compared with the control group at day 7.

Discussion

Immunosuppressants have provided great improvement in organ transplantation by suppressing the rejection of allografts; this has increased the survival rate of organ transplant patients (23). Tacrolimus (FK506) is a widely-used, well-known immunosuppressant used following kidney or heart transplantation, and has been recognized as effective in promoting the growth of bone grafts (24). The present study was performed in order to investigate the effects of tacrolimus on proliferation and osteoblastic differentiation of mesenchymal stem cells in vitro.

This study determined the effects of tacrolimus on the morphology, cell viability and mineralization following treatment with 0.001–100 µg/ml tacrolimus. The cells exposed to tacrolimus at concentrations of 0.001–10 µg/ml exhibited a similar fibroblastic spindle shape. Short-term application of tacrolimus did not result in morphologic changes at final concentrations ranging from 0.001 to 10 µg/ml.

Cellular viability was determined using a CCK-8 assay, which is based on mitochondrial enzyme reduction of the WST-8 and spectrophotometric quantification of the water-soluble formazan dye generated (25). This study showed that tacrolimus at the tested concentrations was not identified to exhibit a significant effect on the viability of stem cells derived from the gingiva at final concentrations ranging from 0.001 to 10 µg/ml. However, tacrolimus at 100 µg/ml decreased cell viability on days 3, 5 and 7.

Osteogenic differentiation can be evaluated by Alizarin red S staining (26). The presence of calcium in cellular deposits was confirmed by Alizarin Red S staining with cetylpyridinium chloride for quantification (27). Tacrolimus at 0.001, 0.01 and 1 µg/ml resulted in the highest degree of mineralized nodule formation on day 7. A previous study showed that tacrolimus at 0.04 and 0.4 µg/ml enhanced osteoblastic differentiation of rat mesenchymal stem cells (20). Co-stimulation with tacrolimus (1.0 µg/ml) and bone morphogenetic protein-9 (100 ng/ml) induced marked osteoblastic differentiation of dedifferentiated fat cells, which were isolated from mature adipocytes using the ceiling culture method and exhibit similar characteristics to mesenchymal stem cells (28). In the present study, the highest differentiation was achieved at 0.01 µg/ml; however, a previous study demonstrated that remarkable osteoblast differentiation was achieved at 1.0 µg/ml (20). The differences may be explained by the different type and stage of the cells, the culturing time period and the culture system (29,30).

A previous study demonstrated that tacrolimus promoted the early stage of osteoinduction (24). The mechanism underlying osteogenic differentiation has not yet been fully elucidated; however, it has been suggested that the osteogenic effect of tacrolimus may involve bone morphogenetic protein signaling (16). A previous report suggested that tacrolimus promoted osteogenic differentiation by activating BMP receptors through interacting with FK506-binding protein 12 (16). Tacrolimus enhanced the positive effects of bone morphogenetic proteins on alikaline phosphatase activity and osteocalcin in mRNA (19). The effects of the combination treatment with bone morphogenetic protein and tacrolimus acted in a dose- and time-dependent manner (19).

In conclusion, the present study demonstrated that treatment with tacrolimus at the tested concentrations ranging from 0.001 to 10 µg/ml did not result in significant changes in the viability of stem cells derived from gingiva, but increased osteogenic differentiation of the stem cells. Further studies related to this phenomenon in an in vivo model are necessary in order to ascertain greater understanding.

Acknowledgments

This study was supported by the 2015 Yeungnam University, Basic Science Research Program (grant nos. 2014R1A1A1002536 and 2015R1A2A2A04005616) and the Medical Research Center Program (grant no. 2015R1A5A2009124) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning.

References