투고일：2017. 5. 8         심사일：2017. 6. 19         게재확정일：2017. 7. 28

아스코르브산의 3차원 줄기세포 배양체에 대한
증식 및 분화 효과 분석

1)가톨릭대학교 의과대학 치과학교실(치주과), 2)프로디젠
이 현 진1), 여 성 일2), 박 준 범1)

ABSTRACT
The effects of ascorbic acid on the morphology and cell proliferation
of three-dimensional stem cell spheroids

1)Department of Periodontics, College of Medicine, The Catholic University,
2)Prodizen Inc
Hyunjin Lee1), Seong-Il Yeo2), Jun-Beom Park1)

Purpose: The effects of various concentrations of ascorbic acid on stem cell spheroids derived from intraoral areas are not known yet. Thus, the purpose of this study is to evaluate the effects of different concentrations of ascorbic acid on the morphology and cellular viability of stem cell spheroids derived from the gingival tissues.

Materials and Methods: Stem cells were plated onto silicon elastomer-based concave microwells and grown in the presence of ascorbic acid at concentrations ranging from 0.003% to 0.3%. The morphology of the cells was viewed under an inverted microscope at day 1, 2, 3 and 5. Qualitative live/dead assay and quantitative cellular viability using Cell Counting Kit-8 were performed on day 2 and day 5.

Results: Gingiva-derived stem cells formed spheroids irrespective of ascorbic acid concentration in silicon elastomer-based concave microwells. Increase in the diameter of spheroid were seen with higher concentrations of ascorbic acid. Higher cellular viability was seen in higher concentrations of ascorbic acid.

Conclusion: Within the experimental setting, the application of ascorbic acid on stem-cell spheroids produced an increase in the size and higher viability with higher dosage. It can be suggested ascorbic acid be applied with stem cell spheroids for tissue engineering purposes.

Key words : ascorbic acid; cell proliferation; cell survival; cellular spheroids; gingiva; stem cells

Corresponding Author
Jun-Beom Park, DDS, MSD, PhD
Department of Periodontics, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea
222 Banpo-daero, Seocho-gu, Seoul, 06591, Republic of Korea
E-mail : jbassoonis@yahoo.co.kr, Tel : +82-10- 4325-2651, Fax : +82-2- 537-2374

This research was supported by Catholic Institute of Cell Therapy (CIC, Seoul, Korea).

Ⅰ. INTRODUCTION
Ascorbic acid is the active component of vitamin C, and it is reported to be a naturally occurring organic compound with antioxidant properties1~2). Ascorbic acid is reported to be an important co-substrate of a large class of enzymes and regulates gene expression by interacting with important transcription factors3). It is shown to play an important role in all stressful conditions linked to inflammatory processes and involve immunity3).

Ascorbic acid is shown to play a role in cell proliferation. A new class of antiproliferative molecules were suggested to come from ascorbic acid derivatives4). High concentrations of L-ascorbic acid specifically inhibit the growth of human leukemic cells via downregulation of hypoxia-inducible factor-1α transcription5). Ascorbic acid and retinoic acid acted synergistically in inhibiting human breast cancer cell proliferation6). It was shown ascorbic acid may involve the expression of genes that induce differentiation and block proliferation and the up-regulation of antioxidant enzymes and proteins involved in apoptosis, cell cycle regulation and DNA repair6~7).
Ascorbic acid is shown to be associated with the differentiation of the tested cells2, 8, 9). Ascorbic acid is an inhibitor of pre-adipocyte cell line differentiation with a dose-dependent effect2). Conversely, ascorbic acid was known to favor the expression of the osteoblastic phenotype in several bone cell systems8). The effects of ascorbic acid on pre-osteoblast gene expression were tested, and ascorbic acid was shown to be essential for the proliferation and differentiation of preosteoblast9). Three-dimensional culture systems have been applied for the evaluation of the effects of the applied agents and they have various advantages over two-dimensional culture systems10~12). Cells in three-dimensional cultures can grow in all directions, mimicking the characteristics in vivo tissue10) The effects of various concentrations of ascorbic acid on stem cell spheroids derived from intraoral areas are not known yet. Thus, the purpose of this study is to evaluate the effects of different concentrations of ascorbic acid on the morphology and cellular viability of stem cell spheroids derived from the gingival tissues.

Ⅱ. MATERIALS AND METHODS

1) Isolation and culture of human gingiva-derived stem cells

Gingival tissues were collected from healthy patients undergoing clinical crown lengthening procedures. The design of the study was reviewed and approved by the Institutional Review Board of the Catholic University of Korea? College of Medicine(no. KC11SIS I0348). Informed consent was obtained from all patients according to the Act on Legal Codes for Biomedical Ethics and Safety and the Declaration of Helsinki. Human gingiva-derived stem cells were isolated and cultivated following the protocol published in the author? previous study13).

The gingival tissues were collected and contained in sterile phosphate-buffered saline(PBS; Welgene, Inc., Gyeongsan-si, Gyeongsangbuk-do, Korea) that included 100 U/mL penicillin and 100 ㎍/ml streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) at 4좧. The tissues were de-epithelialized, separated into 1-2 mm2 fragments, digested in 0.2 ㎛ filtered and modified in a media containing dispase(1 mg/ml; Sigma-Aldrich Co.) and collagenase type IV(2 mg/ml; Sigma-Aldrich Co.). The cell suspension was filtered with a 70 ㎛ cell strainer(Falcon, BD Biosciences, Franklin Lakes, NJ, USA), and the cells were incubated at 37좧 in a humidified incubator with 5% CO2. After 24 hours, the non-adherent cells were washed with phosphate-buffered saline(Welgene, Daegu, South Korea). Fresh media was replaced every two to three days.

2) Formation of spheres and evaluation of cellular morphology

Cells were plated onto the silicon elastomer-based concave microwells(H389600, StemFIT 3D; MicroFIT, Seongnam, Korea) with 600㎛ at a density of 4 x 105 cells/each well and cultured in the media (alpha-minimal essential medium(α-MEM, Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS, Gibco), 200 mM L-glutamine(Sigma-Aldrich Co.), 10 mM of ascorbic acid 2-phosphate(Sigma-Aldrich)) in the presence of ascorbic acid(Sigma-Aldrich Co.) at final concentrations of 0.003%(x 1), 0.03%(x 10), and 0.3%(x 100) (Fig. 1). On day 2, 3 and 5, inverted microscopy(CKX41SF, Olympus Corporation, Tokyo, Japan) was used to evaluate the morphology of the tested stem cells.

3) Determination of cell viability

Spheroid viability was qualitatively analyzed by the Live/Dead Kit assay(Molecular Probes, Eugene, OR, USA). The assay is based on the principle that the activity of intracellular esterase causes non-fluorescent, cell-permeant calcein AM to become intensely fluorescent, giving the viable spheroids an intense, uniform, green fluorescence. Ethidium homodimer enters the damaged cell membrane and then binds to nucleic acids, thereby producing a red fluorescence in the dead cells.

The spheroids were washed twice with PBS, followed by suspension in 1 mL of α-MEM (Gibco) containing 2㎕ of 50mM calcein acetoxymethyl ester working solution and 4㎕ of the 2 mM ethidium homodimer-1 for 15 min at room temperature. In this way, the spheroids were stained with calcein acetoxymethyl ester and ethidium homodimer-1and spheroids were observed under a fluorescence microscope (Axiovert 200; Zeiss, Germany) and confocal laser microscope(LSM800 w/Airyscan, Carl Zeiss, Germany) at day 2 and 5.

4) Determination of cell viability

The cell viability analysis was performed on day 1, 2 and 5. Tetrazolium monosodium salt(Cell Counting Kit-8; CCK-8, Dojindo, Tokyo, Japan) was added to the culture, and the cells were incubated for 2 h at 37좧. The spectrophotometric absorbance at 450 nm was measured for the evaluation of formazan product using a microplate reader(BioTek, Winooski, VT, USA). The tests were performed three times.

5) Statistical analysis

The data were shown as means ± standard deviations of the experiments. A test of normality was performed, and a one-way analysis of variance with post hoc Tukey? test was performed to determine the differences between the groups using a commercially available program(SPSS 12 for Windows, SPSS Inc., Chicago, IL, USA). The level of significance was considered 0.05.

Ⅲ. RESULTS

1) Evaluation of cell morphology and cellular viability

Gingiva-derived stem cells formed spheroids irrespective of ascorbic acid concentration in silicon elastomer-based concave microwells(Fig. 1). The morphology of the stem cell spheroids at day 1 is shown in Figures 2A-2C. The morphology of the spheroids at day 2 was similar to that of day 1(Figs. 2D-2F). There were no significant changes in the morphology with the longer incubation time(Figs. 2G-2L).

2) Qualitative determination of cell viability

The results determined by fluorescent microscope are shown in Figure 3. Most of the cells in the spheroids emitted green fluorescence, and the morphology was round without significant changes on day 2 and day 5. The results obtained under confocal microscope is shown in Figures 4 and 5. The results reveal a clear image of live and dead assay. The green fluorescence showed more intense assay with a higher concentration of ascorbic acid on day 2. However, the red fluorescence was also higher with an increase of the ascorbic acid concentration. The similar trends were seen in the results at day 5.

3) Diameter of stem cell spheroids and quantitative analysis of cell viability

The stem cell spheroid diameter results are seen in Figure 6. The average spheroid diameters of the x 1, x 10 and x 100 groups at day 2 were 615.7 ± 147.4 ㎛, 651.0 ± 127.5 ㎛, and 694.2 ± 86.4 ㎛, respectively. The diameters of the x 1, x 10 and x 100 groups at day 5 were 623.6 ± 151.1 ㎛, 631.6 ± 134.9 ㎛ and 690.7 ± 86.4 ㎛, respectively. There were increasing diameters with higher concentrations of ascorbic acid; however, this did not reach statistical significance (P > 0.05).
The results of cell viability using CCK-8 after culturing at day 1, 2, and 5 are shown in Figure 7. The viability values of the x 1, x 10 and x 100 groups at day 1 were 0.318 ± 0.019, 0.300 ± 0.058 and 3.484 ± 0.303, respectively (P < 0.05). The viability values of the x 1, x 10 and x 100 groups at day 2 were 0.350 ± 0.019, 0.507 ± 0.025 and 2.374 ± 0.141, respectively (P < 0.05). The results at day 5 were 0.276 ± 0.013, 2.483 ± 0.006 and 2.067 ± 0.062, respectively (P < 0.05).

Ⅳ. DISCUSSION
This report discusses the effects of different concentrations of ascorbic acid on the morphology and cellular viability of stem cell spheroids derived from the gingival tissues.

Ascorbic acid used in the test seemed to affect the morphology of spheroids stem cells. An increase in the diameter of the spheroids was noted with a higher concentration of ascorbic acid within the tested concentration and qualitative CCK-8 results showed higher mitochondrial activity with a higher concentration of ascorbic acid. In the previous report, the addition of ascorbic acid resulted in cell viability increase8). The dosage of ascorbic acid varies among different studies5, 8, 14, 15). The dosage used in this study corresponds to 0.16 mM, 1.6 mM and 16 mM. Previous studies testing the effects of ascorbic acid on osteoblast cells used the concentration of 284 ㎛8). In another report, the researchers suggested the recommended dosage of 50 ㎛ for the osteogenic differentiation of bone marrow derived stem cells15). It should be noted the use of higher doses of ascorbic acid may yield a negative effect on cell viability5). However, there could be dose variations used to achieve the maximal effect, depending on the system model, the stage of differentiation of stem cells and the culturing time period14, 16).

Many ways have been suggested for making the cell spheroids17~20). The hanging drop method is one of the most widely used for the fabrication of three-dimensional spheroids21). Another bioreactor method-having a rotating platform-can be applied for cell spheroids18). In this report, the silicon elastomer-based concave microwells were used for the fabrication of stem cell spheroids. Microwell culture systems were suggested to produce spheroids in the defined size with homogenous colonies22). Microwells having a poly(ethylene glycol) surface were reported to generate retrievable cell colonies with controlled sizes and shapes with high viability20).
Cells in spheroids show different characteristics over two-dimensional monolayer cultures11). These spheroids are shown to interact with their surroundings in all three dimensions better than in two-dimension culture systems12). It has also been suggested three-dimensional cultures show higher stability and higher resemblance to human tissue, and this may lead to less use of animal models for the experiments11). Moreover, cell spheroids, when applied in cell therapy, may have greater advantages. Stem cell spheroids are reported to show longer stemness maintenance when compared with two-dimensional cultures23). Stem cell spheroids are shown to secrete significantly higher amounts of growth factors, including vascular endothelial growth factor A24). These three-dimensional spheroids can be maintained without routine passaging and manipulation25, 26). In another study, alkaline phosphatase activities were significantly higher in the three-dimensional cultures when compared with the two-dimensional cultures17, 27). Additionally, these stem cell spheroids can be applied in regeneration medicine without the aid of a scaffold with a higher cell number22, 28).

Various tissues can be used for the candidate of stem cells, including bone marrow, adipose tissue and peripheral blood29, 30). Bone marrow is a reasonable source for stem cells, but the procedure for obtaining stem cells may lead to higher pain and morbility29). However, the intraoral area is a more attractive source for mesenchymal stem cells because such a procedure can be performed under local anesthesia31). The gingival tissue can be obtained during routine daily practices with less limitations for the supply32, 33).

Ⅴ. CONCLUSION

The application of ascorbic acid on stem-cell spheroids produced an increase in the size and higher viability with higher dosage within the experimental setting. It can be suggested ascorbic acid be applied with stem cell spheroids for tissue engineering purposes.

참고문헌
1. Kim DO, Lee KW, Lee HJ, Lee CY. Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J Agric Food Chem 2002;50(13):3713-3717.
2. Rahman F, Al Frouh F, Bordignon B, et al. Ascorbic acid is a dose-dependent inhibitor of adipocyte differentiation, probably by reducing cAMP pool. Front Cell Dev Biol 2014;2:29.
3. Sorice A, Guerriero E, Capone F, Colonna G, Castello G, Costantini S. Ascorbic acid: its role in immune system and chronic inflammation diseases. Mini Rev Med Chem 2014;14(5):444-452.
4. Bordignon B, Chiron J, Fontes M. Ascorbic acid derivatives as a new class of antiproliferative molecules. Cancer Lett 2013;338(2):317-327.
5. Kawada H, Kaneko M, Sawanobori M, et al. High concentrations of L-ascorbic acid specifically inhibit the growth of human leukemic cells via downregulation of HIF-1alpha transcription. PLoS One 2013;8(4):e62717.
6. Kim KN, Pie JE, Park JH, Park YH, Kim HW, Kim MK. Retinoic acid and ascorbic acid act synergistically in inhibiting human breast cancer cell proliferation. J Nutr Biochem 2006;17(7):454-462.
7. Belin S, Kaya F, Duisit G, Giacometti S, Ciccolini J, Fontes M. Antiproliferative effect of ascorbic acid is associated with the inhibition of genes necessary to cell cycle progression. PLoS One 2009;4(2):e4409.
8. Park JB. The effects of dexamethasone, ascorbic acid, and beta-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression. J Surg Res 2012;173(1):99-104.
9. Carinci F, Pezzetti F, Spina AM, et al. Effect of Vitamin C on pre-osteoblast gene expression. Arch Oral Biol 2005;50(5):481-496.
10. Li H, Dai Y, Shu J, Yu R, Guo Y, Chen J. Spheroid cultures promote the stemness of corneal stromal cells. Tissue Cell 2015;47(1):39-48.
11. Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 2015;16(3):5517-5527.
12. Jiang CF, Hsu SH, Tsai KP, Tsai MH. Segmentation and tracking of stem cells in time lapse microscopy to quantify dynamic behavioral changes during spheroid formation. Cytometry A 2015;87(6):491-502.
13. Jin SH, Lee JE, Yun JH, Kim I, Ko Y, Park JB. Isolation and characterization of human mesenchymal stem cells from gingival connective tissue. J Periodontal Res 2015;50(4):461-467.
14. Langenbach F, Handschel J. Effects of dexamethasone, ascorbic acid and beta-glycerophosphate on the osteogenic differentiation of stem cells in vitro. Stem Cell Res Ther 2013;4(5):117.
15. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64(2):295-312.
16. Park JB, Zhang H, Lin CY, et al. Simvastatin maintains osteoblastic viability while promoting differentiation by partially regulating the expressions of estrogen receptors alpha. J Surg Res 2012;174(2):278-283.
17. Shen FH, Werner BC, Liang H, et al. Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation. Spine J 2013;13(1):32-43.
18. Cha HM, Kim SM, Choi YS, Kim DI. Scaffold-free three-dimensional culture systems for mass production of periosteum-derived progenitor cells. J Biosci Bioeng 2015;120(2):218-222.
19. Hsiao C, Palecek SP. Microwell regulation of pluripotent stem cell self-renewal and differentiation. Bionanoscience 2012;2(4):266-276.
20. Karp JM, Yeh J, Eng G, et al. Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells. Lab Chip 2007;7(6):786-794.
21. Bartosh TJ, Ylostalo JH. Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique. Curr Protoc Stem Cell Biol 2014;28:Unit 2B.6.
22. Lee SI, Yeo SI, Kim BB, Ko Y, Park JB. Formation of size-controllable spheroids using gingiva-derived stem cells and concave microwells: Morphology and viability tests. Biomed Rep 2016;4(1):97-101.
23. Zhang S, Liu P, Chen L, Wang Y, Wang Z, Zhang B. The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials 2015;41:15-25.
24. Santos JM, Camoes SP, Filipe E, et al. Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing. Stem Cell Res Ther 2015;6:90.
25. Seymour T, Twigger AJ, Kakulas F. Pluripotency Genes and Their Functions in the Normal and Aberrant Breast and Brain. Int J Mol Sci 2015;16(11):27288-27301.
26. Wei J, Han J, Zhao Y, et al. The importance of three-dimensional scaffold structure on stemness maintenance of mouse embryonic stem cells. Biomaterials 2014;35(27):7724-7733.
27. Singhatanadgit W, Varodomrujiranon M. Osteogenic potency of a 3-dimensional scaffold-free bonelike sphere of periodontal ligament stem cells in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol 2013;116(6):e465-472.
28. Knight E, Przyborski S. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat 2014.
29. Park JB, Bae SS, Lee PW, et al. Comparison of stem cells derived from periosteum and bone marrow of jaw bone and long bone in rabbit models. Tissue Eng Regen Med 2012;9(4):224-230.
30. Park JB, Lee KS, Lee W, Kim HS, Lee KH, Kim IS. Establishment of the chronic bone defect model in experimental model mandible and evaluation of the efficacy of the mesenchymal stem cells in enhancing bone regeneration. Tissue Eng Regen Med 2013;10(1):18-24.
31. Sunil P, Manikandhan R, Muthu M, Abraham S. Stem cell therapy in oral and maxillofacial region: An overview. J Oral Maxillofac Pathol 2012;16(1):58-63.
32. Park JB. Restoration of the severely decayed tooth using crown lengthening with simultaneous tooth-preparation. Eur J Dent 2010;4(2):197-201.
33. Park JB. Root coverage with 2 connective tissue grafts obtained from the same location using a single-incision technique. Quintessence Int 2009;40(5):371-376.

Fig. 1. Schematic illustration of stem cell spheroid fabrication with gingival-derived stem cells with different ascorbic concentrations.
Fig. 2. The morphology of the stem cell spheroids at day 1, 2, 3 and 5. The scale bar indicates 200 μm.
 (A) Group x 1 at day 1 (original magnification x 200).
 (B) Group x 10 at day 1 (original magnification x 200).
 (C) Group x 100 at day 1 (original magnification x 200).
 (D) Group x 1 at day 2 (original magnification x 200).
 (E) Group x 10 at day 2 (original magnification x 200).
 (F) Group x 100 at day 2 (original magnification x 200).
 (G) Group x 1 at day 3 (original magnification x 200).
 (H) Group x 10 at day 3 (original magnification x 200).
 (I) Group x 100 at day 3 (original magnification x 200).
 (J) Group x 1 at day 5 (original magnification x 200).
 (K) Group x 10 at day 5 (original magnification x 200).
 (L) Group x 100 at day 5 (original magnification x 200).
Fig. 3. Live/dead cell image of spheroids at day 2 and day 5 under fluorescent microscope. The scale bar indicates 500 μm.
 (A) Group x 1 at day 2 (original magnification x 100).
 (B) Group x 10 at day 2 (original magnification x 100).
 (C) Group x 100 at day 2 (original magnification x 100).
 (D) Group x 1 at day 5 (original magnification x 100).
 (E) Group x 10 at day5 (original magnification x 100).
 (F) Group x 100 at day 5 (original magnification x 100).
Fig. 4. Qualitative results of cellular viability at day 2 under confocal microscope. Live image, dead image, merged image and central images are provided. The scale bar indicates 100 μm.
 (A-D) Group x 1 (original magnification x 100).
 (E-H) Group x 10 (original magnification x 100).
 (I-L) Group x 100 (original magnification x 100).
Fig. 5. Qualitative results of cellular viability at day 5 under confocal microscope. Live image, dead image, merged image and central images are provided. The scale bar indicates 100 μm.
 (A-D) Group x 1 (original magnification x 100).
 (E-H) Group x 10 (original magnification x 100).
 (I-L) Group x 100 (original magnification x 100).
Fig. 6. The diameter of the spheroids at day 2 and day 5.
Fig. 7. Cellular viability on day 1, 2 and 5.
 * Statistically significant differences were noted when compared with group x 1 and x 10 at day 1.
 ** Statistically significant differences were noted when compared with group x 1 and x 10 at day 2.
 ** Statistically significant differences were noted when compared with group x 1 at day 5.
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