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Use of Human Gingival Fibroblasts for Pre-Vascularization Strategies in Oral Tissue Engineering

Abstract

Background:

Cocultures of human gingival fibrobasts (hGF) and endothelial cells could enhance regeneration and repair models as well as improve vascularization limitations in tissue engineering. The aim of this study was to assess if hGF could support formation of stable vessel-like networks.

Methods:

Explant primary hGF were isolated from gum surgical wastes collected from healthy patients with no history of periodontitis. Human umbilical vein endothelial cells (HUVEC) were two-dimensional (2D) and three-dimensional (3D) cocultured in vitro with hGF at a cell ratio of 1:1 and medium of 1:1 of their respective media during at least 31 days. Vessel quantification of HUVEC networks was performed. In order to investigate the pericyte-like properties of hGF, the expression of perivascular markers α-SMA, NG2, CD146 and PDGFR-β was studied using immunocytochemistry and flow cytometry on 2D cultures.

Results:

hGF were able to support a long-lasting HUVEC network at least 31 days, even in the absence of a bioreactor with flow. As observed, HUVEC started to communicate with each other from day 7, constructing a network. Their interconnection increased significantly between day 2 and day 21 and lasted beyond the 31 days of observation. Moreover, we tried to explain the stability of the networks obtained and showed that a small population of hGF in close vicinity of HUVEC networks expressed perivascular markers.

Conclusion:

These findings highlight a new interesting property concerning hGF, accentuating their relevance in tissue engineering and periodontal regeneration. These promising results need to be confirmed using more 3D applications and in vivo testing.

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References

  1. Rouwkema J, Koopman B, Blitterswijk C, Dhert W, Malda J. Supply of nutrients to cells in engineered tissues. Biotechnol Genet Eng Rev. 2010;26:163–78.

    CAS  Article  Google Scholar 

  2. Smirani R, Rémy M, Devillard R, Naveau A. Engineered prevascularization for oral tissue grafting: a systematic review. Tissue Eng Part B Rev. 2020;26:383–98.

    Article  Google Scholar 

  3. Loibl M, Binder A, Herrmann M, Duttenhoefer F, Richards RG, Nerlich M, et al. Direct cell-cell contact between mesenchymal stem cells and endothelial progenitor cells induces a pericyte-like phenotype in vitro. Biomed Res Int. 2014;2014:395781.

  4. Yeasmin S, Ceccarelli J, Vigen M, Carrion B, Putnam AJ, Tarle SA, et al. Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells. Tissue Eng Part A. 2014;20:1188–96.

    CAS  Article  Google Scholar 

  5. Wei W, An Y, An Y, Fei D, Wang Q. Activation of autophagy in periodontal ligament mesenchymal stem cells promotes angiogenesis in periodontitis. J Periodontol. 2018;89:718–27.

    CAS  Article  Google Scholar 

  6. Iwasaki K, Komaki M, Yokoyama N, Tanaka Y, Taki A, Kimura Y, et al. Periodontal ligament stem cells possess the characteristics of pericytes. J Periodontol. 2013;84:1425–33.

    CAS  Article  Google Scholar 

  7. Bae YK, Kim GH, Lee JC, Seo BM, Joo KM, Lee G, et al. The significance of SDF-1α-CXCR4 axis in in vivo angiogenic ability of human periodontal ligament stem cells. Mol Cells. 2017;40:386–92.

    CAS  Article  Google Scholar 

  8. Cheung JW, Jain D, McCulloch CA, Santerre JP. Pro-angiogenic character of endothelial cells and gingival fibroblasts cocultures in perfused degradable polyurethane scaffolds. Tissue Eng Part A. 2015;21:1587–99.

    CAS  Article  Google Scholar 

  9. Kumar GS. Orban's oral histology and embryology. 13rd ed. Elsevier; 2011.

  10. Fawzy El-Sayed KM, Dörfer CE. Gingival mesenchymal stem/progenitor cells: a unique tissue engineering gem. Stem Cells Int. 2016;2016:7154327.

    Article  Google Scholar 

  11. Matsuda Y, Takahashi K, Kamioka H, Naruse K. Human gingival fibroblast feeder cells promote maturation of induced pluripotent stem cells into cardiomyocytes. Biochem Biophys Res Commun. 2018;503:1798–804.

    CAS  Article  Google Scholar 

  12. Larjava H, Wiebe C, Gallant-Behm C, Hart DA, Heino J, Häkkinen L. Exploring scarless healing of oral soft tissues. J Can Dent Assoc. 2011;77:b18.

    PubMed  Google Scholar 

  13. Ahangar P, Mills SJ, Smith LE, Gronthos S, Cowin AJ. Human gingival fibroblast secretome accelerates wound healing through anti-inflammatory and pro-angiogenic mechanisms. NPJ Regen Med. 2020;5:24.

    CAS  Article  Google Scholar 

  14. Fournier BPJ, Larjava H, Häkkinen L. Gingiva as a source of stem cells with therapeutic potential. Stem Cells Dev. 2013;22:3157–77.

    Article  Google Scholar 

  15. Fournier BPJ, Ferre FC, Couty L, Lataillade JJ, Gourven M, Naveau A, et al. Multipotent progenitor cells in gingival connective tissue. Tissue Eng Part A. 2010;16:2891–9.

    Article  Google Scholar 

  16. Häkkinen L, Larjava H, Fournier BPJ. Distinct phenotype and therapeutic potential of gingival fibroblasts. Cytotherapy. 2014;16:1171–86.

    Article  Google Scholar 

  17. Chiquet M, Katsaros C, Kletsas D. Multiple functions of gingival and mucoperiosteal fibroblasts in oral wound healing and repair. Periodontol 2000. 2015;68:21–40.

    Article  Google Scholar 

  18. Isaac J, Nassif A, Asselin A, Taïhi I, Fohrer-Ting H, Klein C, et al. Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing. Biomaterials. 2018;172:41–53.

    CAS  Article  Google Scholar 

  19. Um Min Allah N, Berahim Z, Ahmad A, Kannan TP. Biological interaction between human gingival fibroblasts and vascular endothelial cells for angiogenesis: a co-culture perspective. Tissue Eng Regen Med. 2017;14:495–505.

    CAS  Article  Google Scholar 

  20. Bordenave L, Baquey C, Bareille R, Lefebvre F, Lauroua C, Guerin V, et al. Endothelial cell compatibility testing of three different Pellethanes. J Biomed Mater Res. 1993;27:1367–81.

    CAS  Article  Google Scholar 

  21. Thébaud NB, Aussel A, Siadous R, Toutain J, Bareille R, Montembault A, et al. Labeling and qualification of endothelial progenitor cells for tracking in tissue engineering: an in vitro study. Int J Artif Organs. 2015;38:224–32.

    Article  Google Scholar 

  22. Kérourédan O, Hakobyan D, Rémy M, Ziane S, Dusserre N, Fricain JC, et al. In situ prevascularization designed by laser-assisted bioprinting: effect on bone regeneration. Biofabrication. 2019;11:045002.

  23. Magnan L, Labrunie G, Marais S, Rey S, Dusserre N, Bonneu M, et al. Characterization of a cell-assembled extracellular matrix and the effect of the devitalization process. Acta Biomater. 2018;82:56–67.

    CAS  Article  Google Scholar 

  24. Carpentier G, Berndt S, Ferratge S, Rasband W, Cuendet M, Uzan G, et al. Angiogenesis analyzer for ImageJ—A comparative morphometric analysis of “Endothelial Tube Formation Assay” and “Fibrin Bead Assay.” Sci Rep. 2020;10:11568.

    CAS  Article  Google Scholar 

  25. Woloszyk A, Buschmann J, Waschkies C, Stadlinger B, Mitsiadis TA. Human dental pulp stem cells and gingival fibroblasts seeded into silk fibroin scaffolds have the same ability in attracting vessels. Front Physiol. 2016;7:140.

    PubMed  PubMed Central  Google Scholar 

  26. Costa-Almeida R, Gomez-Lazaro M, Ramalho C, Granja PL, Soares R, Guerreiro SG. Fibroblast-endothelial partners for vascularization strategies in tissue engineering. Tissue Eng Part A. 2015;21:1055–65.

    CAS  Article  Google Scholar 

  27. Alfonso García SL, Parada-Sanchez MT, Arboleda Toro D. The phenotype of gingival fibroblasts and their potential use in advanced therapies. Eur J Cell Biol. 2020;99:151123.

    Article  Google Scholar 

  28. Herndon JM, Tome ME, Davis TP. Development and maintenance of the blood–brain barrier. In: Primer on Cerebrovascular Diseases. Academic Press; 2017. p. 51–6.

  29. Dore-Duffy P, Cleary K. Morphology and properties of pericytes. Methods Mol Biol. 2011;686:49–68.

    CAS  Article  Google Scholar 

  30. Crisan M, Corselli M, Chen WC, Péault B. Perivascular cells for regenerative medicine. J Cell Mol Med. 2012;16:2851–60.

    CAS  Article  Google Scholar 

  31. Ozerdem U, Stallcup WB. Pathological angiogenesis is reduced by targeting pericytes via the NG2 proteoglycan. Angiogenesis. 2004;7:269–76.

    CAS  Article  Google Scholar 

  32. Wang Z, Xu Q, Zhang N, Du X, Xu G, Yan X. CD146, from a melanoma cell adhesion molecule to a signaling receptor. Signal Transduct Target Ther. 2020;5:148.

    CAS  Article  Google Scholar 

  33. Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, et al. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development. Proc Natl Acad Sci U S A. 2017;114:E7622–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Suphasiriroj W, Mikami M, Sato S. Comparative studies on microvascular endothelial cells isolated from periodontal tissue. J Periodontol. 2013;84:1002–9.

    CAS  Article  Google Scholar 

  35. Liu X, Wang J, Dong F, Li H, Hou Y. Human gingival fibroblasts induced and differentiated into vascular endothelial-like cells. Dev Growth Differ. 2016;58:702–13.

    CAS  Article  Google Scholar 

  36. Girolamo F, de Trizio I, Errede M, Longo G, d’Amati A, Virgintino D. Neural crest cell-derived pericytes act as pro-angiogenic cells in human neocortex development and gliomas. Fluids Barriers CNS. 2021;18:14.

    Article  Google Scholar 

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Acknowledgements

This work was supported by a grant from the Fondation de l’Avenir pour la Recherche Médicale [AP-RM-20-025]. The authors would like to thank Hugo Oliveira (ART BioPrint, Bordeaux) for providing the collagen/hyaluronic acid biomaterial ink for 3D coculture.

Funding

Fondation de l'Avenir pour la Recherche Médicale Appliquée, AP-RM-20-025, Adrien Naveau.

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Correspondence to Rawen Smirani.

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Ethical statement

Samples were harvested according to the French legislation i.e. under the control of the declaration for conservation and preparation of human body elements for scientific research number DC 2008-412 (French ministry of higher education, research and innovation). Protocols were approved by the institutional committee for the protection of human subjects (Local Ethic Committee from academic hospital CHU de Bordeaux).

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Smirani, R., Rémy, M., Devillard, R. et al. Use of Human Gingival Fibroblasts for Pre-Vascularization Strategies in Oral Tissue Engineering. Tissue Eng Regen Med 19, 525–535 (2022). https://doi.org/10.1007/s13770-021-00415-3

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  • DOI: https://doi.org/10.1007/s13770-021-00415-3

Keywords

  • Tissue engineering
  • Fibroblast
  • Endothelial cell
  • Coculture techniques
  • Pericyte