Influence of Hypoxic Condition on Cytotoxicity, Cellular Migration, and Osteogenic Differentiation Potential of Aged Periodontal Ligament Cells

Abstract

Objective This study aimed to investigate and compare the influence of hypoxic conditions on cytotoxicity, cellular migration, and osteogenic differentiation of aged periodontal ligament (PDL) cells.

Materials and Methods Isolated human PDL cells from aged and young subjects were cultured under hypoxic conditions, which were treated with hydrogen peroxide (H₂O₂) (0, 25, 50, 100, 200, and 500 µM). To assess cytotoxicity, lactate dehydrogenase release was determined by the optical density at 490 nm, and the percentage of cell death was calculated. An in vitro wound healing assay was performed over 24 to 48 hours for cellular migration determination. Osteogenic differentiation was determined by alizarin red staining and osteogenic gene expression, including the expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN).

Results There was a significant difference in the percentage of cell death with high hypoxic condition (200 and 500 µM) compared to low hypoxic conditions on both day 1 and 2. The highest cellular migration was depicted at 50 µM in both young and aged groups of the in vitro wound healing assay. Osteogenic gene expression of RUNX2 in the aged group was increased at 25 and 50 µM hypoxic condition at day 7, but the expression was gradually decreased after 14 days. On the contrary, the expression of ALP and OPN in the aged group was increased at day 14. Only OPN had been found to be statistically significantly different when compared with gene expression at day 7 and 14 (p < 0.05). The results showed no statistically significant differences when compared with the young and aged groups in all genes and all concentrations.

Conclusion The concentration of low hypoxic condition (25–50 µM) was proposed to promote cell viability, cellular migration, and osteogenic differentiation in aged PDL cells. We suggested that the potential of aged PDL cells for use in cell therapy for periodontal regeneration might possibly be similar to that of young PDL cells.

Keywords

periodontal ligament - cell hypoxia - cell migration assays - osteogenesis - cell differentiation

Publikationsverlauf

Artikel online veröffentlicht:
17. Mai 2024

• References

• 1 Hajishengallis G, Chavakis T, Lambris JD. Current understanding of periodontal disease pathogenesis and targets for host-modulation therapy. Periodontol 2000 2020; 84 (01) 14-34
• 2 Cekici A, Kantarci A, Hasturk H, Van Dyke TE. Inflammatory and immune pathways in the pathogenesis of periodontal disease. Periodontol 2000 2014; 64 (01) 57-80
• 3 Swoboda JR, Kiyak HA, Darveau R, Persson GR. Correlates of periodontal decline and biologic markers in older adults. J Periodontol 2008; 79 (10) 1920-1926
• 4 Yu YH, Kuo HK, Lai YL. The association between serum folate levels and periodontal disease in older adults: data from the National Health and Nutrition Examination Survey 2001/02. J Am Geriatr Soc 2007; 55 (01) 108-113
• 5 Chung HY, Lee EK, Choi YJ. et al. Molecular inflammation as an underlying mechanism of the aging process and age-related diseases. J Dent Res 2011; 90 (07) 830-840
• 6 Fransson C, Mooney J, Kinane DF, Berglundh T. Differences in the inflammatory response in young and old human subjects during the course of experimental gingivitis. J Clin Periodontol 1999; 26 (07) 453-460
• 7 Norderyd O, Hugoson A, Grusovin G. Risk of severe periodontal disease in a Swedish adult population. A longitudinal study. J Clin Periodontol 1999; 26 (09) 608-615
• 8 Ogawa H, Yoshihara A, Hirotomi T, Ando Y, Miyazaki H. Risk factors for periodontal disease progression among elderly people. J Clin Periodontol 2002; 29 (07) 592-597
• 9 Renvert S, Persson GR. Treatment of periodontal disease in older adults. Periodontol 2000 2016; 72 (01) 108-119
• 10 Trombelli L, Rizzi A, Simonelli A, Scapoli C, Carrieri A, Farina R. Age-related treatment response following non-surgical periodontal therapy. J Clin Periodontol 2010; 37 (04) 346-352
• 11 Han NY, Hong JY, Park JM. et al. Label-free quantitative proteomic analysis of human periodontal ligament stem cells by high-resolution mass spectrometry. J Periodontal Res 2019; 54 (01) 53-62
• 12 Xing Y, Zhang Y, Wu X, Zhao B, Ji Y, Xu X. A comprehensive study on donor-matched comparisons of three types of mesenchymal stem cells-containing cells from human dental tissue. J Periodontal Res 2019; 54 (03) 286-299
• 13 Bryant SR, Zarb GA. Osseointegration of oral implants in older and younger adults. Int J Oral Maxillofac Implants 1998; 13 (04) 492-499
• 14 Yeo EJ. Hypoxia and aging. Exp Mol Med 2019; 51 (06) 1-15
• 15 Valli A, Harris AL, Kessler BM. Hypoxia metabolism in ageing. Aging (Albany NY) 2015; 7 (07) 465-466
• 16 Kim JH, Song SY, Park SG, Song SU, Xia Y, Sung JH. Primary involvement of NADPH oxidase 4 in hypoxia-induced generation of reactive oxygen species in adipose-derived stem cells. Stem Cells Dev 2012; 21 (12) 2212-2221
• 17 Busletta C, Novo E, Valfrè Di Bonzo L. et al. Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells. Stem Cells 2011; 29 (06) 952-963
• 18 Li S, Deng Y, Feng J, Ye W. Oxidative preconditioning promotes bone marrow mesenchymal stem cells migration and prevents apoptosis. Cell Biol Int 2009; 33 (03) 411-418
• 19 De Barros S, Dehez S, Arnaud E. et al. Aging-related decrease of human ASC angiogenic potential is reversed by hypoxia preconditioning through ROS production. Mol Ther 2013; 21 (02) 399-408
• 20 Kim JH, Park SG, Song SY, Kim JK, Sung JH. Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2. Cell Death Dis 2013; 4 (04) e588
• 21 Hung SP, Ho JH, Shih YRV, Lo T, Lee OK. Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells. J Orthop Res 2012; 30 (02) 260-266
• 22 Kifune T, Ito H, Ishiyama M. et al. Hypoxia-induced upregulation of angiogenic factors in immortalized human periodontal ligament fibroblasts. J Oral Sci 2018; 60 (04) 519-525
• 23 Ng KT, Li JP, Ng KM, Tipoe GL, Leung WK, Fung ML. Expression of hypoxia-inducible factor-1α in human periodontal tissue. J Periodontol 2011; 82 (01) 136-141
• 24 Zou D, Han W, You S. et al. In vitro study of enhanced osteogenesis induced by HIF-1α-transduced bone marrow stem cells. Cell Prolif 2011; 44 (03) 234-243
• 25 Nagatomo K, Komaki M, Sekiya I. et al. Stem cell properties of human periodontal ligament cells. J Periodontal Res 2006; 41 (04) 303-310
• 26 Jung GA, Kim JA, Park HW. et al. Induction of Nanog in neural progenitor cells for adaptive regeneration of ischemic brain. Exp Mol Med 2022; 54 (11) 1955-1966
• 27 De Miguel MP, Alcaina Y, de la Maza DS, Lopez-Iglesias P. Cell metabolism under microenvironmental low oxygen tension levels in stemness, proliferation and pluripotency. Curr Mol Med 2015; 15 (04) 343-359
• 28 Morimoto C, Takedachi M, Kawasaki K. et al. Hypoxia stimulates collagen hydroxylation in gingival fibroblasts and periodontal ligament cells. J Periodontol 2021; 92 (11) 1635-1645
• 29 Ji G, Lv K, Chen H. et al. MiR-146a regulates SOD2 expression in H2O2 stimulated PC12 cells. PLoS One 2013; 8 (07) e69351
• 30 Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 2005; 11 (06) 990-995
• 31 Chen Z, Patwari M, Liu D. Cytotoxicity of orthodontic temporary anchorage devices on human periodontal ligament fibroblasts in vitro. Clin Exp Dent Res 2019; 5 (06) 648-654
• 32 Ye J, Tang X, Long Y, Chu Z, Zhou Q, Lin B. The effect of hypoxia on the proliferation capacity of dermal papilla cell by regulating lactate dehydrogenase. J Cosmet Dermatol 2021; 20 (02) 684-690
• 33 Wang G, Chen S, Shao Z. et al. Metformin alleviates hydrogen peroxide-induced inflammation and oxidative stress via inhibiting P2 × 7R signaling in spinal cord tissue cells neurons. Can J Physiol Pharmacol 2021; 99 (08) 768-774
• 34 Potier E, Ferreira E, Andriamanalijaona R. et al. Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. Bone 2007; 40 (04) 1078-1087
• 35 Aziz NSM, Stanslas J, Kadir NH ABD. Hydrogen peroxide promotes human skin fibroblast cells migration and proliferation at lower dose via VEGF-alpha and TGF-alpha gene expression. Malays Appl Biol 2020; 49: 261-266
• 36 Iida K, Takeda-Kawaguchi T, Tezuka Y, Kunisada T, Shibata T, Tezuka K. Hypoxia enhances colony formation and proliferation but inhibits differentiation of human dental pulp cells. Arch Oral Biol 2010; 55 (09) 648-654
• 37 Pitkänen S. In Vitro and In Vivo Osteogenesis and Vasculogenesis in Synthetic Bone Grafts [thesis]. Tampere, Finland: Tampere University; 2020: 120p
• 38 Grynpas M. Age and disease-related changes in the mineral of bone. Calcif Tissue Int 1993; 53 (Suppl. 01) S57-S64
• 39 Kaplan SS, Kaplan T, Sezgin GP. Assessment of the frequency and correlation of carotid artery calcifications and pulp stones with idiopathic osteosclerosis using digital panoramic radiographs. Clin Exp Health Sci 2021; 11: 342-347
• 40 Hung SC, Pochampally RR, Hsu SC. et al. Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo. PLoS One 2007; 2 (05) e416
• 41 Chow DC, Wenning LA, Miller WM, Papoutsakis ET. Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models. Biophys J 2001; 81 (02) 685-696
• 42 Ferrer A, Roser CT, El-Far MH. et al. Hypoxia-mediated changes in bone marrow microenvironment in breast cancer dormancy. Cancer Lett 2020; 488: 9-17
• 43 Hubbi ME, Semenza GL. Regulation of cell proliferation by hypoxia-inducible factors. Am J Physiol Cell Physiol 2015; 309 (12) C775-C782