Abstract
Aim: The aim of this study was to compare dental pulp tissue in human exfoliated deciduous teeth (SHEDs) and dental pulp stem cells (DPSCs) in response to ascorbic acid as the sole osteoblast inducer.
Background: A cocktail of ascorbic acid, β-glycerophosphate, and dexamethasone has been widely used to induce osteoblast differentiation. However, under certain conditions, β-glycerophosphate and dexamethasone can cause a decrease in cell viability in stem cells.
Objectives: This study aims to determine the cytotoxic effect and potential of ascorbic acid as the sole inducer of osteoblast differentiation.
Methods: Cytotoxicity analyses in the presence of 10-500 μg/mL ascorbic acid were performed in both cell types using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The concentrations below the IC50 (i.e., 10-150 μg/mL) were used to determine osteoblast differentiation potential of ascorbic acid using the alkaline phosphatase (ALP) assay, von Kossa staining, and reverse transcription-polymerase chain reaction.
Results: SHEDs and DPSCs proliferated for 21 days, expressed a Mesenchymal Stem Cell (MSC) marker (CD73+), and did not express Hematopoietic Stem Cell (HSC) markers (CD34- and SLAMF1-). SHEDs had a higher range of IC50 values (215-240 μg/mL ascorbic acid), while the IC50 values for DPSCs were 177-211 μg/mL after 24-72 hours. SHEDs treated with 10-100 μg/mL ascorbic acid alone exhibited higher ALP-specific activity and a higher percentage of mineralisation than DPSCs. Both cell types expressed osteoblast markers on day 21, i.e., RUNX2+ and BSP+, in the presence of ascorbic acid.
Conclusions: SHEDs survive at higher concentrations of ascorbic acid as compared to DPSC. The cytotoxic effect was only exhibited at ≥250 μg/mL ascorbic acid. In addition, SHED exhibited better ALP and mineralization activities, but lower osteoblast marker expression than DPSC in response to ascorbic acid as the sole inducer.
Keywords: Comparative, ascorbic acid, cytotoxicity, osteoblast differentiation, SHED, DPSC.
Graphical Abstract
[1]
Ansari AS, Yazid MD, Sainik NQAV, Razali RA, Saim AB, Idrus RBH. Osteogenic induction of wharton’s jelly-derived mesenchymal stem cell for bone regeneration: A systematic review. Stem Cells Int 2018; 2018: 2406462.
[http://dx.doi.org/10.1155/2018/2406462] [PMID: 30534156]
[http://dx.doi.org/10.1155/2018/2406462] [PMID: 30534156]
[2]
Ishikawa S, Iwasaki K, Komaki M, Ishikawa I. Role of ascorbic acid in periodontal ligament cell differentiation. J Periodontol 2004; 75(5): 709-16.
[http://dx.doi.org/10.1902/jop.2004.75.5.709] [PMID: 15214312]
[http://dx.doi.org/10.1902/jop.2004.75.5.709] [PMID: 15214312]
[3]
Kunimatsu R, Nakajima K, Awada T, et al. Comparative characterization of stem cells from human exfoliated deciduous teeth, dental pulp, and bone marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun 2018; 501(1): 193-8.
[http://dx.doi.org/10.1016/j.bbrc.2018.04.213] [PMID: 29730288]
[http://dx.doi.org/10.1016/j.bbrc.2018.04.213] [PMID: 29730288]
[4]
Wu Y-K, Tu Y-K, Yu J, Cheng N-C. The influence of cell culture density on the cytotoxicity of adipose-derived stem cells induced by l-ascorbic acid-2-phosphate. Sci Rep 2020; 10(1): 104.
[http://dx.doi.org/10.1038/s41598-019-56875-0] [PMID: 31919399]
[http://dx.doi.org/10.1038/s41598-019-56875-0] [PMID: 31919399]
[5]
Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; 81(8): 531-5.
[http://dx.doi.org/10.1177/154405910208100806] [PMID: 12147742]
[http://dx.doi.org/10.1177/154405910208100806] [PMID: 12147742]
[6]
Hu J, Cao Y, Xie Y, et al. Periodontal regeneration in swine after cell injection and cell sheet transplantation of human dental pulp stem cells following good manufacturing practice. Stem Cell Res Ther 2016; 7(1): 130.
[http://dx.doi.org/10.1186/s13287-016-0362-8] [PMID: 27613503]
[http://dx.doi.org/10.1186/s13287-016-0362-8] [PMID: 27613503]
[7]
Tomlinson MJ, Dennis C, Yang XB, Kirkham J. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture. Cell Tissue Res 2015; 361(2): 529-40.
[http://dx.doi.org/10.1007/s00441-014-2106-3] [PMID: 25636587]
[http://dx.doi.org/10.1007/s00441-014-2106-3] [PMID: 25636587]
[8]
Zainal Ariffin SH, Kermani S, Zainol Abidin IZ, et al. Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium. Stem Cells Int 2013; 2013: 250740.
[http://dx.doi.org/10.1155/2013/250740] [PMID: 24348580]
[http://dx.doi.org/10.1155/2013/250740] [PMID: 24348580]
[9]
Zainal Ariffin SH, Kermani S, Megat Abdul Wahab R, Senafi S, Zainal Ariffin Z, Abdul Razak M. In vitro chondrogenesis transformation study of mouse dental pulp stem cells. Sci World J 2012; 2012: 827149.
[http://dx.doi.org/10.1100/2012/827149] [PMID: 22919354]
[http://dx.doi.org/10.1100/2012/827149] [PMID: 22919354]
[10]
Ariffin SH, Manogaran T, Abidin IZ, Wahab RM, Senafi S. A perspective on stem cells as biological systems that produce differentiated osteoblasts and odontoblasts. Curr Stem Cell Res Ther 2017; 12(3): 247-59.
[http://dx.doi.org/10.2174/1574888X11666161026145149] [PMID: 27784228]
[http://dx.doi.org/10.2174/1574888X11666161026145149] [PMID: 27784228]
[11]
Kerkis I, Kerkis A, Dozortsev D, et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs 2006; 184(3-4): 105-16.
[http://dx.doi.org/10.1159/000099617] [PMID: 17409736]
[http://dx.doi.org/10.1159/000099617] [PMID: 17409736]
[12]
Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000; 21(11): 1095-102.
[http://dx.doi.org/10.1016/S0142-9612(99)00192-1] [PMID: 10817261]
[http://dx.doi.org/10.1016/S0142-9612(99)00192-1] [PMID: 10817261]
[13]
Ganta DR, McCarthy MB, Gronowicz GA. Ascorbic acid alters collagen integrins in bone culture. Endocrinology 1997; 138(9): 3606-12.
[http://dx.doi.org/10.1210/endo.138.9.5367] [PMID: 9275042]
[http://dx.doi.org/10.1210/endo.138.9.5367] [PMID: 9275042]
[14]
Hadzir SN, Ibrahim SN, Abdul Wahab RM, et al. Ascorbic acid induces osteoblast differentiation of human suspension mononuclear cells. Cytotherapy 2014; 16(5): 674-82.
[http://dx.doi.org/10.1016/j.jcyt.2013.07.013] [PMID: 24176546]
[http://dx.doi.org/10.1016/j.jcyt.2013.07.013] [PMID: 24176546]
[15]
Schäck LM, Noack S, Winkler R, et al. The phosphate source influences gene expression and quality of mineralization during in vitro osteogenic differentiation of human mesenchymal stem cells. PLoS One 2013; 8(6): e65943.
[http://dx.doi.org/10.1371/journal.pone.0065943] [PMID: 23823126]
[http://dx.doi.org/10.1371/journal.pone.0065943] [PMID: 23823126]
[16]
Orriss IR, Hajjawi MO, Huesa C, MacRae VE, Arnett TR. Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. Int J Mol Med 2014; 34(5): 1201-8.
[http://dx.doi.org/10.3892/ijmm.2014.1926] [PMID: 25200658]
[http://dx.doi.org/10.3892/ijmm.2014.1926] [PMID: 25200658]
[17]
Orriss IR, Taylor SE, Arnett TR. Rat osteoblast cultures. Bone research
protocols. Totowa, NJ: Humana Press 2012; pp. 31-41.
[18]
Patel JJ, Bourne LE, Davies BK, et al. Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts. Exp Cell Res 2019; 380(1): 100-13.
[http://dx.doi.org/10.1016/j.yexcr.2019.04.020] [PMID: 31004580]
[http://dx.doi.org/10.1016/j.yexcr.2019.04.020] [PMID: 31004580]
[19]
Kim SM, Kim YG, Park JW, Lee JM, Suh JY. The effects of dexamethasone on the apoptosis and osteogenic differentiation of human periodontal ligament cells. J Periodontal Implant Sci 2013; 43(4): 168-76.
[http://dx.doi.org/10.5051/jpis.2013.43.4.168] [PMID: 24040569]
[http://dx.doi.org/10.5051/jpis.2013.43.4.168] [PMID: 24040569]
[20]
Postnov A, De Schutter T, Sijbers J, Karperien M, De Clerck N. Glucocorticoid-induced osteoporosis in growing mice is not prevented by simultaneous intermittent PTH treatment. Calcif Tissue Int 2009; 85(6): 530-7.
[http://dx.doi.org/10.1007/s00223-009-9301-3] [PMID: 19844646]
[http://dx.doi.org/10.1007/s00223-009-9301-3] [PMID: 19844646]
[21]
Li J, Zhang N, Huang X, Xu J, Fernandes JC, Dai K, et al. Dexamethasone shifts bone marrow stromal cells from osteoblasts to adipocytes by C/EBPalpha promoter methylation. Cell Death Dis 2013; 4(10): e832-e.
[22]
Megat Abdul Wahab R, Mohamed Rozali NA, Senafi S, Zainol Abidin IZ, Zainal Ariffin Z, Zainal Ariffin SH. Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells. PeerJ 2017; 5: e3180.
[http://dx.doi.org/10.7717/peerj.3180] [PMID: 28626603]
[http://dx.doi.org/10.7717/peerj.3180] [PMID: 28626603]
[23]
Zainal Ariffin SH, Mohamed Rozali NA, Megat Abdul Wahab R, Senafi S, Zainol Abidin IZ, Zainal Ariffin Z. Analyses of basal media and serum for in vitro expansion of suspension peripheral blood mononucleated stem cell. Cytotechnology 2016; 68(4): 675-86.
[http://dx.doi.org/10.1007/s10616-014-9819-8] [PMID: 26231833]
[http://dx.doi.org/10.1007/s10616-014-9819-8] [PMID: 26231833]
[24]
Hagar MN, Yazid F, Luchman NA, Ariffin SHZ, Wahab RMA. Comparative evaluation of osteogenic differentiation potential of stem cells derived from dental pulp and exfoliated deciduous teeth cultured over granular hydroxyapatite based scaffold. BMC Oral Health 2021; 21(1): 263.
[http://dx.doi.org/10.1186/s12903-021-01621-0] [PMID: 33992115]
[http://dx.doi.org/10.1186/s12903-021-01621-0] [PMID: 33992115]
[25]
Reisman M, Adams KT. Stem cell therapy: A look at current research, regulations, and remaining hurdles. PT 2014; 39(12): 846-57.
[PMID: 25516694]
[PMID: 25516694]
[26]
Ariffin SHZ, Abidin IZZ, Yazid MD, Wahab RMA. Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus. Cell Commun Signal 2010; 8: 29.
[http://dx.doi.org/10.1186/1478-811X-8-29] [PMID: 20969794]
[http://dx.doi.org/10.1186/1478-811X-8-29] [PMID: 20969794]
[27]
Intan Zarina ZA, Shahrul Hisham ZA, Rohaya MAW, Sahidan S, Zaidah ZA. Osteoclast and osteoblast development of mus musculus haemopoietic mononucleated cells. J Biol Sci 2008; 8: 506-16.
[http://dx.doi.org/10.3923/jbs.2008.506.516]
[http://dx.doi.org/10.3923/jbs.2008.506.516]
[28]
D’Aniello C, Cermola F, Patriarca EJ, Minchiotti G. Vitamin C in stem cell biology: impact on extracellular matrix homeostasis and epigenetics. Stem Cells Int 2017; 2017: 8936156.
[http://dx.doi.org/10.1155/2017/8936156] [PMID: 28512473]
[http://dx.doi.org/10.1155/2017/8936156] [PMID: 28512473]
[29]
Hasmad H, Yusof MR, Mohd Razi ZR, Hj Idrus RB, Chowdhury SR. Human amniotic membrane with aligned electrospun fiber as scaffold for aligned tissue regeneration. Tissue Eng Part C Methods 2018; 24(6): 368-78.
[http://dx.doi.org/10.1089/ten.tec.2017.0447] [PMID: 29690856]
[http://dx.doi.org/10.1089/ten.tec.2017.0447] [PMID: 29690856]
[30]
Rojas A, Aguilar R, Henriquez B, et al. Epigenetic control of the bone-master runx2 gene during osteoblast-lineage commitment by the histone demethylase JARID1B/KDM5B. J Biol Chem 2015; 290(47): 28329-42.
[http://dx.doi.org/10.1074/jbc.M115.657825] [PMID: 26453309]
[http://dx.doi.org/10.1074/jbc.M115.657825] [PMID: 26453309]
[31]
Kirches E, Steffen T, Waldt N, et al. The expression of the MSC- marker CD73 and of NF2/Merlin are correlated in meningiomas. J Neurooncol 2018; 138(2): 251-9.
[http://dx.doi.org/10.1007/s11060-018-2807-7] [PMID: 29468444]
[http://dx.doi.org/10.1007/s11060-018-2807-7] [PMID: 29468444]
[32]
Monguió-Tortajada M, Roura S, Gálvez-Montón C, Franquesa M, Bayes-Genis A, Borràs FE. Mesenchymal stem cells induce expression of CD73 in human monocytes in vitro and in a swine model of myocardial infarction in vivo. Front Immunol 2017; 8: 1577.
[http://dx.doi.org/10.3389/fimmu.2017.01577] [PMID: 29209319]
[http://dx.doi.org/10.3389/fimmu.2017.01577] [PMID: 29209319]
[33]
Ab Kadir R, Ariffin SH, Wahab RM, Senafi S, Huyop FZ. Differentiation potential of human suspension mononucleated peripheral blood cells. Afr J Biotechnol 2011; 10(52): 10756-64.
[http://dx.doi.org/10.5897/AJB11.436]
[http://dx.doi.org/10.5897/AJB11.436]
[34]
Hartmann K, Koenen M, Schauer S, et al. Molecular actions of glucocorticoids in cartilage and bone during health, disease, and steroid therapy. Physiol Rev 2016; 96(2): 409-47.
[http://dx.doi.org/10.1152/physrev.00011.2015] [PMID: 26842265]
[http://dx.doi.org/10.1152/physrev.00011.2015] [PMID: 26842265]
[35]
Blair HC, Larrouture QC, Li Y, et al. Osteoblast differentiation and bone matrix formation in vivo and in vitro. Tissue Eng Part B Rev 2017; 23(3): 268-80.
[http://dx.doi.org/10.1089/ten.teb.2016.0454] [PMID: 27846781]
[http://dx.doi.org/10.1089/ten.teb.2016.0454] [PMID: 27846781]
[36]
Li JW, Xu C, Fan Y, Wang Y, Xiao YB. Can serum levels of alkaline phosphatase and phosphate predict cardiovascular diseases and total mortality in individuals with preserved renal function? A systemic review and meta-analysis. PLoS One 2014; 9(7): e102276.
[http://dx.doi.org/10.1371/journal.pone.0102276] [PMID: 25033287]
[http://dx.doi.org/10.1371/journal.pone.0102276] [PMID: 25033287]
[37]
Kermani S, Megat Abdul Wahab R, Zarina Zainol Abidin I, Zainal Ariffin Z, Senafi S, Hisham Zainal Ariffin S. Differentiation capacity of mouse dental pulp stem cells into osteoblasts and osteoclasts. Cell J 2014; 16(1): 31-42.
[PMID: 24518973]
[PMID: 24518973]
[38]
Shiga M, Kapila YL, Zhang Q, Hayami T, Kapila S. Ascorbic acid induces collagenase-1 in human periodontal ligament cells but not in MC3T3-E1 osteoblast-like cells: Potential association between collagenase expression and changes in alkaline phosphatase phenotype. J Bone Mineral Res 2003; 18(1): 67-77.
[http://dx.doi.org/10.1359/jbmr.2003.18.1.67]
[http://dx.doi.org/10.1359/jbmr.2003.18.1.67]
[39]
Intan Zarina ZA, Shahrul Hisham ZA, Zaidah ZA, Rohaya MAW. Potential differentiation of three types of primitive cells originated from different proliferation terms of mouse blood. Sains Malays 2010; 39(2): 305-13.
[40]
Akiva A, Malkinson G, Masic A, et al. On the pathway of mineral deposition in larval zebrafish caudal fin bone. Bone 2015; 75: 192-200.
[http://dx.doi.org/10.1016/j.bone.2015.02.020] [PMID: 25725266]
[http://dx.doi.org/10.1016/j.bone.2015.02.020] [PMID: 25725266]
[41]
Boonrungsiman S, Gentleman E, Carzaniga R, et al. The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc Natl Acad Sci USA 2012; 109(35): 14170-5.
[http://dx.doi.org/10.1073/pnas.1208916109] [PMID: 22879397]
[http://dx.doi.org/10.1073/pnas.1208916109] [PMID: 22879397]
[42]
Wang H, Zhong Q, Yang T, et al. Comparative characterization of SHED and DPSCs during extended cultivation in vitro. Mol Med Rep 2018; 17(5): 6551-9.
[http://dx.doi.org/10.3892/mmr.2018.8725] [PMID: 29532869]
[http://dx.doi.org/10.3892/mmr.2018.8725] [PMID: 29532869]
[43]
Wang S, Noda K, Yang Y, Shen Z, Chen Z, Ogata Y. Calcium hydroxide regulates transcription of the bone sialoprotein gene via a calcium-sensing receptor in osteoblast-like ROS 17/2.8 cells. Eur J Oral Sci 2018; 126(1): 13-23.
[http://dx.doi.org/10.1111/eos.12392] [PMID: 29159829]
[http://dx.doi.org/10.1111/eos.12392] [PMID: 29159829]
[44]
Yang Y, Huang Y, Zhang L, Zhang C. Transcriptional regulation of bone sialoprotein gene expression by Osx. Biochem Biophys Res Commun 2016; 476(4): 574-9.
[http://dx.doi.org/10.1016/j.bbrc.2016.05.164] [PMID: 27261434]
[http://dx.doi.org/10.1016/j.bbrc.2016.05.164] [PMID: 27261434]
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