Potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus

The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres were prepared using electrospraying technique. In vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (pH 7.4). Gingiva-derived stem cells were isolated and incubated with tacrolimus or tacrolimus-loaded microspheres. Release study of the microspheres revealed prolonged release profiles of tacrolimus without any significant initial burst release. The microsphere itself did not affect the morphology of the mesenchymal stem cells, and cell morphology was retained after incubation with microspheres loaded with tacrolimus at 1 μg/mL to 10 μg/mL. Cultures grown in the presence of microspheres loaded with tacrolimus at 1 μg/mL showed the highest mineralization. Alkaline phosphatase activity increased with an increase in incubation time. The highest expression of pSmad1/5 was achieved in the group receiving tacrolimus 0.1 μg/mL every third day, and the highest expression of osteocalcin was achieved in the group receiving 1 μg/mL every third day. Biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus promoted mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation.

Differentiation of stem cell is significant in bone tissue engineering because stem cells are considered as attractive cell sources for bone tissue engineering 1,2 . It was shown that the delivery of agents including drugs and growth factors can enhance the differentiation ability 3,4 . In this study, biodegradable microspheres were applied for the sustained delivery of drugs for the enhancement of the differentiation ability human mesenchymal stem cells.
Tacrolimus is a potent macrolide lactone immunosuppressive agent used for prophylaxis of organ rejection after transplantation and graft-versus-host disease after bone marrow transplantation in patients 5 . Tacrolimus is known to decrease the action of the immune system and this may increase the risk of viral infection 6 . Tacrolimus exerts a variety of actions on bone metabolism 4,7,8 . The osteogenic differentiation of bone marrow-derived mesenchymal stem cells is greatly promoted by the application of tacrolimus 7 . In vivo osteogenic capability of cultured cells in porous hydroxyapatite using a rat model showed increased bone formation with tacrolimus 8 . Similarly, our previous report showed that tacrolimus in concentrations ranging from 0.001 to 10 μ g/mL did not produce statistically significant differences in the viability of stem cells derived from gingiva, but rather enhanced the osteogenic differentiation of the stem cells 4 .
A previous report showed that tacrolimus-loaded biodegradable microspheres achieved sustained release over a long period, yielding flat parallel concentration profiles for 10 days from the first day after a single subcutaneous administration in liver-transplanted rats 9 .
The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. In vitro release study. The in vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (PBS, pH 7.4; 1% tween 20) at 37 °C in a shaking incubator. Briefly, 1 mg equivalent of tacrolimus was taken in triplicate, and a suspension was made in a small volume of release medium. The suspension was then loaded in a dialysis membrane with molecular weight cut-off 3.5 kDa, and the membrane was clipped on both sides to ensure no leakage took place. The membrane was then kept in a tube containing 10 mL of release medium and incubated at the predetermined conditions. At predetermined time intervals, sampling was performed, and the whole of the release medium was replaced with an equivalent amount of fresh medium. The sampling was performed at two-day interval starting at day 1 to day 21. Two additional sampling was performed at day 25 and day 30. The release samples were analyzed by high-performance liquid chromatography analysis.
Alizarin Red S staining. Cell cultures grown with osteogenic media were obtained on days 7 and 14, washed twice with PBS (Welgene), fixed with 70% ethanol, and rinsed twice with deionized water. Cultures were stained with Alizarin Red S for 30 minutes at room temperature. To remove non-specifically bound stain, cultures were washed three times with deionized water and once with PBS for 15 minutes at ambient temperature. Bound dye was solubilized in 10 mM sodium phosphate containing 10% cetylpyridinium chloride and quantified spectrophotometrically at 560 nm. (C) Quantitative results of mineralization assay on days 7 and 14. *Statistically significant differences noted versus control on day 7. **Statistically significant differences noted when compared with TO 1 on day 7. # Significant differences were noted versus TO 10 group on day 7. ## Significant differences were noted versus T3/1 group on day 7. † Significant differences were noted versus T3/10 group on day 7. † † Significant differences were noted versus MS group on day 7. ‡ Significant differences were noted versusTM0.1 group on day 7. ‡ ‡ Significant differences were noted versus TM1 group on day 7. ‖ Significant differences were noted versus control on day 7. ‖‖ Significant differences were noted versus control on day 14.

Statistical analysis.
The data are presented as means ± standard deviations of the experiments. One-way analysis of variance (ANOVA) with post hoc test was performed to determine the differences between groups using a commercially available program (SPSS 12 for Windows, SPSS Inc., Chicago, IL, USA). The level of significance was 0.05.

Characterization of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres. Scanning elec-
tron microscopy. Figure 1 shows the scanning electron microscopy of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres. As revealed from the microscopy, homogenously size-distributed, drug-loaded microspheres were obtained by electrospraying. The diameter of microspheres ranged from 3 to 6 μ m (Fig. 1A,B). Because of the Coulomb fission that occurs during the evaporation of organic solvent, a few small particles were also observed (arrow).
Encapsulation efficiency and loading capacity. The encapsulation efficiency and loading capacity were 90.77 ± 0.95% and 9.41 ± 0.41%, respectively. Due to the sufficiently hydrophobic nature of tacrolimus, it was well incorporated inside the PLGA microspheres, leading to good encapsulation efficiency.
In vitro release study. The release study of the microspheres revealed prolonged release profile of tacrolimus, extending to more than 25 days without a significant initial burst release (Fig. 1C). A high degree of correlation (r 2 = 0.9728) to zero order equation (Q o -Q t = K o t, where 'Q' is the amount of drug dissolved in time 't' , 'Q o ' is the initial amount of drug in the solution, and 'K o ' is the zero order rate constant) was found, indicating that the drug release is independent of the concentration of loaded drug in the microspheres. Moreover, the release profile also showed good correlation (r 2 = 0.9897) with Korsmeyer-Peppas model (M t /M ∞ = K.t n , where 'M t /M ∞ ' is the fraction of drug released after time 't' , 'K' is the release constant and 'n' is the release exponent, which characterizes the different release mechanisms), indicating that diffusion was the primary mechanism involved in the drug release from the formulation. Also, the n value in Korsmeyer-Peppas model suggested that the mechanism of drug release is related to drug diffusion as well as polymer degradation.
The morphology of the cells on day 5 is shown in Fig. 3. The shapes of the cells in the tested groups were similar to the control group except for the 100 μ g/mL group. Cellular viability. The CCK-8 results on days 2 and 5 are shown in Fig. 4. Compared to the control, growth in the presence of tacrolimus at 100 μ g/mL resulted in decreases in the CCK-8 values on days 2 and 5, respectively (P < 0.05). All groups except the unloaded control, the 100 μ g/mL group, and microspheres loaded with tacrolimus at 10 μ g/mL showed statistically significant increases in cell proliferation over time.
Mineralization assay. Mineralized extracellular deposits were minimally observed after Alizarin Red S staining on days 7 and 14 (Fig. 5A,B). Increase of mineralized deposits was noted on day 14 when compared with day 7. The quantitative results regarding bound dye on days 7 and 14 are shown in Fig. 5C.
Cultures grown in the presence of 1 μ g/mL and 10 μ g/mL tacrolimus; T3/0.1 group, T3/1 group and T3/10 group; and TM10 group showed statistically significant increases of mineralized deposits compared with the control on day 7.
The results for day 14 showed that treatment with tacrolimus generally increased deposits compared with those on day 7 in each group. Cultures grown in the presence of 10 μ g/mL tacrolimus, groups of 1 μ g/mL changed with fresh media containing tacrolimus every third day, and microspheres loaded with tacrolimus at 0.1, 1, and 10 μ g/mL showed statistically significant increases compared with the control on day 14.
The differences in the alkaline phosphatase activity between the groups did not reach statistical significance. However, a statistically significant decrease was observed only between the control group and 100 μ g/mL tacrolimus group on days 5 and 7 (P < 0.05).

Discussion
This report discusses the effects of biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus on the proliferation and differentiation of mesenchymal stem cells derived from the gingiva.
This study used an electrospray technique with a single-nozzle electrospraying machine. A previous study used electrospraying to prepare poly(lactic-co-glycolic acid) microparticles and further encapsulate the drug, and electrospraying was concluded to be a cost-effective, single-step process for the preparation of polymeric microparticles 12 . Scanning electron microscopy revealed a uniform round shape, and loading of tacrolimus did not interfere with microsphere formation. The size and charge of the particles could be controlled by regulating the polymer solution flow rate and electric voltage 13 . This study showed that microsphere formation led to prolonged release of the drug without significant initial burst. Similarly, previous research reported electrosprayed poly(lactic-co-glycolic acid) microdevices for sustained drug delivery for over three weeks 14 . The release profile could be controlled by the drug:polymer ratio, and previous results showed that the drug:polymer ratio of 1:10 had slower release profiles than those with a 1:5 ratio 15 .
Cytotoxicity evaluation of the microsphere itself did not affect the morphology of the mesenchymal stem cells. Moreover, cell morphology was retained after incubation with microspheres loaded with tacrolimus at final concentration of 1 μ g/mL to 10 μ g/mL. However, higher doses of tacrolimus (100 μ g/mL) produced rounder shapes with fewer cells. Cellular viability was evaluated using CCK-8 assay, which is based on mitochondrial enzyme reduction of the water-soluble tetrazolium salt-8-(2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt) and quantification of any generated water-soluble Scientific RepoRts | 6:34910 | DOI: 10.1038/srep34910 formazan 16 . Cellular viability can be evaluated using various methods [17][18][19] . The dye exclusion test is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue 17 . The protein assay is an indirect measurement of cell viability because it measures the protein content of viable cells that are left after washing the treated plates 18,19 . Thus, CCK-8 assay can be regarded as a more sensitive assay because it measures cell viability through the determination of mitochondrial dehydrogenase activity. It should be noted that the use of higher doses of tacrolimus was previously shown to yield a negative effect on cell viability 20 , and our study proved that tacrolimus at 100 μ g/mL decreased cell viability on days 2 and 5.
Following the period of matrix maturation, nodule cells begin to mineralize the extracellular matrix 21 . Alizarin Red S staining was used to evaluate the presence of calcium deposits, and cetylpyridinium chloride was applied for the quantitative analysis 22 . A previous report showed that tacrolimus at 0.04 μ g/mL and 0.4 μ g/mL enhanced osteoblastic differentiation of mesenchymal stem cells 20 . Our previous research showed that tacrolimus showed the highest mineralized nodule formation at 0.001, 0.01, and 1 μ g/mL 4 . In this report, cultures grown in the presence of microspheres loaded with tacrolimus at 1 μ g/mL showed the highest mineralization.
Alkaline phosphatase activity is considered an early marker of osteogenic differentiation 23 . Alkaline phosphatase activities increased between 5 and 7 days, but there were no significant differences noted except for the 100 μ g/mL group. However, higher alkaline phosphatase activity was previously noted in the allografts with tacrolimus 24 . The differences in the responses may be explained by the types and stages of the cells, the culturing time period, and the system 25 .
Western blot analysis was performed to detect protein expression of pSmad1/5 and osteocalcin following treatment with tacrolimus to provide information of additional possible mechanisms. Tacrolimus may influence osteoblast differentiation through bone morphogenetic protein signaling 26 . Highest expression of pSmad1/5 was achieved in the tacrolimus (0.1 μ g/mL) group changed with fresh media containing tacrolimus every third day. A previous report showed that co-stimulation with tacrolimus at 1.0 μ g/mL and bone morphogenetic protein-9 at 100 ng/mL induced remarkable osteoblastic differentiation 27 . Osteocalcin is reported to be an osteoblast-specific gene expressed by fully differentiated osteoblasts 28 . Highest expression of osteocalcin was achieved in the tacrolimus (1 μ g/mL) group changed with fresh media containing tacrolimus every third day, but it did not reach statistical significance.
Based on these findings, biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus produced prolonged release profiles with increased mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation.