CC BY 4.0 · Eur J Dent
DOI: 10.1055/s-0043-1764422
Original Article

Induction of Migration and Collagen Synthesis in Human Gingival Fibroblasts Using Periodontal Ligament Stem Cell Conditioned Medium

Akkapol Banlue
1   Department of Oral Medicine and Periodontology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
Jirattikarn Kaewmuangmoon
2   Department of Anatomy, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
2   Department of Anatomy, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
Kallapat Tansriratanawong
1   Department of Oral Medicine and Periodontology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand
› Author Affiliations
Funding Our research was supported by the National Research Council of Thailand (2020).


Objective This study aimed to examine the effect of periodontal ligament stem cell conditioned medium (PDLSC-CM) on human gingival fibroblast (HGF) migration and collagen synthesis.

Materials and Methods To assess cell viability, we extracted PDLSC-CM, and the total derived protein concentration was adjusted to 12.5 to 200 µg/mL, followed by treatment with HGFs. The viability of HGFs was observed for 24 hours using the MTT assay. Cell migration was monitored for 24 to 48 hours by wound healing and Boyden chamber assays. Collagen synthesis from HGFs was examined by picrosirius red dye and real-time polymerase chain reaction (PCR) to measure collagen type I and III gene expression for 7 to 10 days. A comparison among the groups was assessed using a one-way analysis of variance (ANOVA) and Bonferroni post hoc test, with the exception of the cell viability assay, which was subjected to Welch's test and Dunnett's T3 post hoc test.

Results HGF viability was significantly enhanced by 12.5, 25, and 50 µg/mL PDLSC-CM. The HGFs treated with 50 µg/mL PDLSC-CM promoted cell migration as shown by wound healing and Boyden chamber assays. At this concentration, collagen synthesis increased at 10 days. Collagen type I gene expression increased by 1.6-fold (p < 0.001) and 4.96-fold (p < 0.001) at 7 and 10 days, respectively. Collagen type III gene expression showed an increase of 1.76-fold (p < 0.001) and 6.67-fold (p < 0.001) at the same time points.

Conclusion Our study suggested that a low concentration of PDLSC-CM at 50 µg/mL has given an amelioration of HGFs providing for periodontal wound healing and periodontal regeneration, particularly migration and collagen synthesis.

Ethics Statement

The present study was approved by the Faculty of Dentistry/Faculty of Pharmacy, Mahidol University, Institutional Review Board (MU-DT/PY-IRB 2021/DT093).

Publication History

Article published online:
27 April 2023

© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (

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  • References

  • 1 Caplan AI. The mesengenic process. Clin Plast Surg 1994; 21 (03) 429-435
  • 2 Bartold PM, McCulloch CA, Narayanan AS, Pitaru S. Tissue engineering: a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000 2000; 24: 253-269
  • 3 Zhu W, Liang M. Periodontal ligament stem cells: current status, concerns, and future prospects. Stem Cells Int 2015; 2015: 972313
  • 4 Bartold PM, Shi S, Gronthos S. Stem cells and periodontal regeneration. Periodontol 2000 2006; 40: 164-172
  • 5 Asgarpour K, Shojaei Z, Amiri F. et al. Exosomal microRNAs derived from mesenchymal stem cells: cell-to-cell messages. Cell Commun Signal 2020; 18 (01) 149
  • 6 Pawitan JA. Prospect of stem cell conditioned medium in regenerative medicine. BioMed Res Int 2014; 2014: 965849
  • 7 Nagata M, Iwasaki K, Akazawa K. et al. Conditioned medium from periodontal ligament stem cells enhances periodontal regeneration. Tissue Eng Part A 2017; 23 (9–10): 367-377
  • 8 Qiu J, Wang X, Zhou H. et al. Enhancement of periodontal tissue regeneration by conditioned media from gingiva-derived or periodontal ligament-derived mesenchymal stem cells: a comparative study in rats. Stem Cell Res Ther 2020; 11 (01) 42
  • 9 Novello S, Tricot-Doleux S, Novella A, Pellen-Mussi P, Jeanne S. Influence of periodontal ligament stem cell-derived conditioned medium on osteoblasts. Pharmaceutics 2022; 14 (04) 1-17
  • 10 Tang Y, Zhou Y, Li HJ. Advances in mesenchymal stem cell exosomes: a review. Stem Cell Res Ther 2021; 12 (01) 71
  • 11 Gholami L, Nooshabadi VT, Shahabi S. et al. Extracellular vesicles in bone and periodontal regeneration: current and potential therapeutic applications. Cell Biosci 2021; 11 (01) 16
  • 12 Smith PC, Martínez C, Martínez J, McCulloch CA. Role of fibroblast populations in periodontal wound healing and tissue remodeling. Front Physiol 2019; 10: 270
  • 13 Amzica F, Nanci A. Cytoskeleton, cell junctions, fibroblasts, and extracellular matrix. In: Nanci A. eds. Ten Cate's Oral Histology. 8th ed. St. Louis, MO: Mosby; 2013: 48-69
  • 14 Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res 2010; 89 (03) 219-229
  • 15 Cáceres M, Oyarzun A, Smith PC. Defective wound-healing in aging gingival tissue. J Dent Res 2014; 93 (07) 691-697
  • 16 Swift ME, Kleinman HK, DiPietro LA. Impaired wound repair and delayed angiogenesis in aged mice. Lab Invest 1999; 79 (12) 1479-1487
  • 17 Rangaraj A, Harding K, Leaper D. Role of collagen in wound management. Wounds 2011; 7: 54-63
  • 18 Makrantonaki E, Wlaschek M, Scharffetter-Kochanek K. Pathogenesis of wound healing disorders in the elderly. J Dtsch Dermatol Ges 2017; 15 (03) 255-275
  • 19 Zhao J, Liu D, Duan F, Wei Y. Effect of mesenchymal stem cell conditioned medium on human gingival fibroblast proliferation and collagen synthesis. Int J Clin Exp Med 2017; 10 (11) 15244-15249
  • 20 Seubbuk S, Sritanaudomchai H, Kasetsuwan J, Surarit R. High glucose promotes the osteogenic differentiation capability of human periodontal ligament fibroblasts. Mol Med Rep 2017; 15 (05) 2788-2794
  • 21 Lin H, Chen H, Zhao X. et al. Advances in mesenchymal stem cell conditioned medium-mediated periodontal tissue regeneration. J Transl Med 2021; 19 (01) 456
  • 22 Jantanasan S, Tansriratanawong K, Janebodin K, Kaewmuangmoon J. The proliferative effect of conditioned media from human periodontal ligament stem cells and human exfoliated deciduous teeth stem cells on human gingival fibroblasts. Mahidol Dental Journal 2021; 41 (02) 139-150
  • 23 Aghamohamadi Z, Kadkhodazadeh M, Torshabi M, Tabatabaei F. A compound of concentrated growth factor and periodontal ligament stem cell-derived conditioned medium. Tissue Cell 2020; 65: 101373
  • 24 Daskalaki E, Pillon NJ, Krook A, Wheelock CE, Checa A. The influence of culture media upon observed cell secretome metabolite profiles: the balance between cell viability and data interpretability. Anal Chim Acta 2018; 1037: 338-350
  • 25 Hassell T, Gleave S, Butler M. Growth inhibition in animal cell culture. The effect of lactate and ammonia. Appl Biochem Biotechnol 1991; 30 (01) 29-41
  • 26 Nishimura F, Terranova VP. Comparative study of the chemotactic responses of periodontal ligament cells and gingival fibroblasts to polypeptide growth factors. J Dent Res 1996; 75 (04) 986-992
  • 27 Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2 (02) 329-333
  • 28 Entschladen F, Drell IV TL, Lang K. et al. Analysis methods of human cell migration. Exp Cell Res 2005; 307 (02) 418-426
  • 29 Chen HC. Boyden chamber assay. Methods Mol Biol 2005; 294: 15-22
  • 30 Clark RAF. Overview and general considerations of wound repair. In: Clark RAF, Henson PM. eds. The Molecular and Cellular Biology of Wound Repair. Boston, MA: Springer US; 1988: 3-33
  • 31 Cooper JA, Mintz BR, Palumbo SL, Li WJ. Assays for determining cell differentiation in biomaterials. In: Jaffe M, Hammond W, Tolias P, Arinzeh T. eds. Characterization of Biomaterials. Cambridge, UK: Woodhead Publishing; 2013: 101-137
  • 32 Junquiera LC, Junqueira LC, Brentani RR. A simple and sensitive method for the quantitative estimation of collagen. Anal Biochem 1979; 94 (01) 96-99
  • 33 Yang H, Wang C, Chen H. et al. Neural stem cell-conditioned medium ameliorated cerebral ischemia-reperfusion injury in rats. Stem Cells Int 2018; 2018: 4659159
  • 34 Asgari Taei A, Dargahi L, Nasoohi S, Hassanzadeh G, Kadivar M, Farahmandfar M. The conditioned medium of human embryonic stem cell-derived mesenchymal stem cells alleviates neurological deficits and improves synaptic recovery in experimental stroke. J Cell Physiol 2021; 236 (03) 1967-1979
  • 35 Kim MJ, Kim ZH, Kim SM, Choi YS. Conditioned medium derived from umbilical cord mesenchymal stem cells regenerates atrophied muscles. Tissue Cell 2016; 48 (05) 533-543
  • 36 Park CM, Kim MJ, Kim SM, Park JH, Kim ZH, Choi YS. Umbilical cord mesenchymal stem cell-conditioned media prevent muscle atrophy by suppressing muscle atrophy-related proteins and ROS generation. In Vitro Cell Dev Biol Anim 2016; 52 (01) 68-76
  • 37 Benavides-Castellanos MP, Garzón-Orjuela N, Linero I. Effectiveness of mesenchymal stem cell-conditioned medium in bone regeneration in animal and human models: a systematic review and meta-analysis. Cell Regen (Lond) 2020; 9 (01) 5
  • 38 Katagiri W, Osugi M, Kawai T, Hibi H. First-in-human study and clinical case reports of the alveolar bone regeneration with the secretome from human mesenchymal stem cells. Head Face Med 2016; 12: 5
  • 39 Saheli M, Bayat M, Ganji R. et al. Human mesenchymal stem cells-conditioned medium improves diabetic wound healing mainly through modulating fibroblast behaviors. Arch Dermatol Res 2020; 312 (05) 325-336
  • 40 Hammarström L, Heijl L, Gestrelius S. Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 1997; 24 (9, Pt 2): 669-677
  • 41 Qi L, Liu L, Hu Y. et al. Concentrated growth factor promotes gingival regeneration through the AKT/Wnt/β-catenin and YAP signaling pathways. Artif Cells Nanomed Biotechnol 2020; 48 (01) 920-932
  • 42 Morand DN, Davideau JL, Clauss F, Jessel N, Tenenbaum H, Huck O. Cytokines during periodontal wound healing: potential application for new therapeutic approach. Oral Dis 2017; 23 (03) 300-311
  • 43 Chew JRJ, Chuah SJ, Teo KYW. et al. Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration. Acta Biomater 2019; 89: 252-264
  • 44 Yue C, Cao J, Wong A. et al. human bone marrow stromal cell exosomes ameliorate periodontitis. J Dent Res 2022; 101 (09) 1110-1118