Skip to main content

Advertisement

SpringerLink
Log in

Combined use of hydrogen-rich water and enzyme-digested edible bird’s nest improves PMA/LPS-impaired wound healing in human inflammatory gingival tissue equivalents

  • Research Article
  • Published:
Human Cell Aims and scope Submit manuscript

Abstract

Gingival wound healing plays a critical role in maintaining oral health. However, this process can be delayed by oxidative stress and excessive inflammatory responses. In this study, we established a human inflammatory gingival tissue equivalent (iGTE) to investigate the inhibitory effects of hydrogen-rich water (HW), enzyme-digested edible bird’s nest (EBND) and sialic acid (SA) on PMA (an inducer of oxidative free radicals)- and LPS (an inflammatory stimulus)-impaired wound healing. The iGTE was constructed by human gingival fibroblasts (hGFs), keratinocytes and macrophages under three-dimensional conditions. Wounds in the iGTE and hGF/keratinocyte monolayers were created by mechanical injury. Tissues and cells were pretreated with HW, EBND, and SA, and then exposed to the inflammatory and oxidative environment induced by PMA (10 ng/mL) and LPS (250 ng/mL). The inflammatory cytokines IL-6 and IL-8 were quantitatively analyzed by ELISA. Histopathological image analysis was performed by HE and immunofluorescence staining. In the iGTE, PMA/LPS significantly reduced the epithelial thickness while causing a decrease in K8/18, E-cadherin, laminin and elastin expression and an increase in COX-2 expression along with ulcer-like lesions. In mechanically scratched hGFs and keratinocyte monolayers, PMA/LPS significantly impaired wound healing, and promoted the secretion of IL-6 and IL-8. Pretreatment of HW, EBND, and SA significantly suppressed PMA/LPS-induced wound healing delay and inflammatory responses in cell monolayers, as well as in the iGTE. Remarkably, the combined use of HW and EBND exhibited particularly robust results. Combined use of HW and EBND may be applied for the prevention and treatment of wound healing delay.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The data presented in this study are available within the article. Other data related to this study are available on request from the corresponding author.

References

  1. Smith PC, Cáceres M, Martínez C, Oyarzún A. Martínez J. Gingival wound healing: an essential response disturbed by aging? J Dent Res. 2015;94:395–402. https://doi.org/10.1177/0022034514563750.

  2. 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. https://doi.org/10.1038/s41536-020-00109-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Front Physiol. 2018;9:419. https://doi.org/10.3389/fphys.2018.00419.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Larouche J, Sheoran S, Maruyama K, Martino MM. Immune regulation of skin wound healing: mechanisms and novel therapeutic targets. Adv Wound Care (New Rochelle). 2018;7:209–31. https://doi.org/10.1089/wound.2017.0761.

    Article  PubMed  Google Scholar 

  5. Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res. 2010;89:219–29. https://doi.org/10.1177/0022034509359125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis. 2004;17:91–6. https://doi.org/10.1097/00001432-200404000-00004.

    Article  PubMed  Google Scholar 

  7. Dunnill C, Patton T, Brennan J, et al. Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int Wound J. 2017;14:89–96. https://doi.org/10.1111/iwj.12557.

    Article  PubMed  Google Scholar 

  8. Crompton R, Williams H, Ansell D, et al. Oestrogen promotes healing in a bacterial LPS model of delayed cutaneous wound repair. Lab Invest. 2016;96:439–49. https://doi.org/10.1038/labinvest.2015.160.

    Article  CAS  PubMed  Google Scholar 

  9. Wang G, Yang F, Zhou W, Xiao N, Luo M, Tang Z. The initiation of oxidative stress and therapeutic strategies in wound healing. Biomed Pharmacother. 2023;157: 114004. https://doi.org/10.1016/j.biopha.2022.114004.

    Article  PubMed  Google Scholar 

  10. Cho YD, Kim KH, Lee YM, Ku Y, Seol YJ. Periodontal wound healing and tissue regeneration: a narrative review. Pharmaceuticals (Basel). 2021;14:456. https://doi.org/10.3390/ph14050456.

    Article  CAS  PubMed  Google Scholar 

  11. Sim M, Kim CS, Shon WJ, Lee YK, Choi EY, Shin DM. Hydrogen-rich water reduces inflammatory responses and prevents apoptosis of peripheral blood cells in healthy adults: a randomized, double-blind, controlled trial. Sci Rep. 2020;10:12130. https://doi.org/10.1038/s41598-020-68930-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mikami T, Tano K, Lee H, et al. Drinking hydrogen water enhances endurance and relieves psychometric fatigue: a randomized, double-blind, placebo-controlled study 1. Can J Physiol Pharmacol. 2019;97:857–62. https://doi.org/10.1139/cjpp-2019-0059.

    Article  CAS  PubMed  Google Scholar 

  13. Gu Y, Huang CS, Inoue T, et al. Drinking hydrogen water ameliorated cognitive impairment in senescence-accelerated mice. J Clin Biochem Nutr. 2010;46:269–76. https://doi.org/10.3164/jcbn.10-19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xiao L, Miwa N. Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging and relief from glutathione diminishment. Hum Cell. 2017;30:72–87. https://doi.org/10.1007/s13577-016-0150-x.

    Article  CAS  PubMed  Google Scholar 

  15. Kucerova R, Ou J, Lawson D, Leiper LJ, Collinson JM. Cell surface glycoconjugate abnormalities and corneal epithelial wound healing in the pax6+/− mouse model of aniridia-related keratopathy. Invest Ophthalmol Vis Sci. 2006;47:5276–82. https://doi.org/10.1167/iovs.06-0581.

    Article  PubMed  Google Scholar 

  16. Ghatak S, Maytin EV, Mack JA, et al. Roles of proteoglycans and glycosaminoglycans in wound healing and fibrosis. Int J Cell Biol. 2015;2015: 834893. https://doi.org/10.1155/2015/834893.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yida Z, Imam MU, Ismail M, et al. Edible Bird’s Nest attenuates high fat diet-induced oxidative stress and inflammation via regulation of hepatic antioxidant and inflammatory genes. BMC Complement Altern Med. 2015;15:310. https://doi.org/10.1186/s12906-015-0843-9.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Hu Q, Li G, Yao H, et al. Edible bird’s nest enhances antioxidant capacity and increases lifespan in Drosophila Melanogaster. Cell Mol Biol (Noisy-le-Grand). 2016;62:116–122.

  19. Vimala B, Hussain H, Wan Nazaimoon WM. Effects of edible bird’s nest on tumour necrosis factor-α secretion, nitric oxide production and cell viability of lipopolysaccharide-stimulated RAW 264.7 macrophages. Food Agric Immunol. 2012;23:303–314. https://doi.org/10.1080/09540105.2011.625494.

  20. Chua KH, Mohamed IN, Mohd Yunus MH, et al. The anti-viral and anti-inflammatory properties of edible bird’s nest in influenza and coronavirus infections: from pre-clinical to potential clinical application. Front Pharmacol. 2021;12: 633292. https://doi.org/10.3389/fphar.2021.633292.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang D, Shimamura N, Mochizuki M, Nakahara T, Sunada K, Xiao L. Enzyme-digested edible bird’s nest (EBND) prevents UV and arid environment-induced cellular oxidative stress, cell death and DNA damage in human skin keratinocytes and three-dimensional epithelium equivalents. Antioxidants (Basel). 2023;12:609. https://doi.org/10.3390/antiox12030609.

    Article  CAS  PubMed  Google Scholar 

  22. Xiao L, Miwa N. Hydrogen nano-bubble water suppresses ROS generation, adipogenesis, and interleukin-6 secretion in hydrogen-peroxide- or PMA-stimulated adipocytes and three-dimensional subcutaneous adipose equivalents. Cells. 2021;10:626. https://doi.org/10.3390/cells10030626.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988;106:761–71. https://doi.org/10.1083/jcb.106.3.761.

    Article  CAS  PubMed  Google Scholar 

  24. Xiao L, Mochizuki M, Nakahara T, Miwa N. Hydrogen-generating silica material prevents UVA-ray-induced cellular oxidative stress, cell death, collagen loss and melanogenesis in human cells and 3D skin equivalents. Antioxidants (Basel). 2021;10:76. https://doi.org/10.3390/antiox10010076.

    Article  CAS  PubMed  Google Scholar 

  25. Xiao L, Okamura H, Kumazawa Y. Three-dimensional inflammatory human tissue equivalents of gingiva. J Vis Exp. 2018; 57157. https://doi.org/10.3791/57157

  26. Xiao L, Mochizuki M, Wang D, Shimamura N, Sunada K, Nakahara T. Types of cell culture inserts affect cell crosstalk between co-cultured macrophages and adipocytes. Biochem Biophys Res Commun. 2023;658:10–7. https://doi.org/10.1016/j.bbrc.2023.03.068.

    Article  CAS  PubMed  Google Scholar 

  27. Burgess A, Vigneron S, Brioudes E, Labbé JC, Lorca T, Castro A. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA. 2010;107:12564–9. https://doi.org/10.1073/pnas.0914191107.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Xiao L, Mochizuki M, Fan Y, Nakahara T, Liao F. Enzyme-digested Colla Corii Asini (E’jiao) suppresses lipopolysaccharide-induced inflammatory changes in THP-1 macrophages and OP9 adipocytes. Hum Cell. 2022;35:885–95. https://doi.org/10.1007/s13577-022-00694-5.

    Article  CAS  PubMed  Google Scholar 

  29. Xiao L, Sakagami H, Miwa N. A new method for testing filtration efficiency of mask materials under sneeze-like pressure. In Vivo. 2020;34:1637–44. https://doi.org/10.21873/invivo.11955.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fujita T, Yoshimoto T, Kajiya M, et al. Regulation of defensive function on gingival epithelial cells can prevent periodontal disease. Jpn Dent Sci Rev. 2018;4:66–75. https://doi.org/10.1016/j.jdsr.2017.11.003.

    Article  Google Scholar 

  31. Baechle JJ, Chen N, Makhijani P, Winer S, Furman D, Winer DA. Chronic inflammation and the hallmarks of aging. Mol Metab. 2023;74: 101755. https://doi.org/10.1016/j.molmet.2023.101755.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Claffey N, Shanley D. Relationship of gingival thickness and bleeding to loss of probing attachment in shallow sites following nonsurgical periodontal therapy. J Clin Periodontol. 1986;13:654–7. https://doi.org/10.1111/j.1600-051x.1986.tb00861.x.

    Article  CAS  PubMed  Google Scholar 

  33. Kolte R, Kolte A, Mahajan A. Assessment of gingival thickness with regards to age, gender and arch location. J Indian Soc Periodontol. 2014;18:478–81. https://doi.org/10.4103/0972-124X.138699.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Singh J, Rathod VJ, Rao PR, Patil AA, Langade DG, Singh RK. Correlation of gingival thickness with gingival width, probing depth, and papillary fill in maxillary anterior teeth in students of a dental college in Navi Mumbai. Contemp Clin Dent. 2016;7:535–8. https://doi.org/10.4103/0976-237X.194117.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Kasai Y, Sugiyama H, Takagi R, et al. Brush biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine. Regen Ther. 2016;4:71–7. https://doi.org/10.1016/j.reth.2016.02.008.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Schreurs O, Karatsaidis A, Balta MG, Grung B, Hals EKB, Schenck K. Expression of keratins 8, 18, and 19 in epithelia of atrophic oral lichen planus. Eur J Oral Sci. 2020;128:7–17. https://doi.org/10.1111/eos.12666.

    Article  CAS  PubMed  Google Scholar 

  37. Ernstsen CV, Riishede A, Iversen AKS, Bay L, Bjarnsholt T, Nejsum LN. E-cadherin and aquaporin-3 are downregulated in wound edges of human chronic wounds. APMIS. 2023;131:403–9. https://doi.org/10.1111/apm.13332.

    Article  CAS  PubMed  Google Scholar 

  38. Caley MP, Martins VL, O’Toole EA. Metalloproteinases and Wound Healing. Adv Wound Care (New Rochelle). 2015;4:225–34. https://doi.org/10.1089/wound.2014.0581.

    Article  PubMed  Google Scholar 

  39. Woo SH, Choi JH, Mo YJ, Lee YI, Jeon WB, Lee YS. Engineered elastin-like polypeptide improves the efficiency of adipose-derived stem cell-mediated cutaneous wound healing in type II diabetes mellitus. Heliyon. 2023;9: e20201. https://doi.org/10.1016/j.heliyon.2023.e20201.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chen MR, Dragoo JL. The effect of nonsteroidal anti-inflammatory drugs on tissue healing. Knee Surg Sports Traumatol Arthrosc. 2013;21:540–9. https://doi.org/10.1007/s00167-012-2095-2.

    Article  PubMed  Google Scholar 

  41. Masson-Meyers DS, Andrade TAM, Caetano GF, et al. Experimental models and methods for cutaneous wound healing assessment. Int J Exp Pathol. 2020;101:21–37. https://doi.org/10.1111/iep.12346.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Smith CJ, Parkinson EK, Yang J, et al. Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures. J Dent. 2022;125: 104251. https://doi.org/10.1016/j.jdent.2022.104251.

    Article  CAS  PubMed  Google Scholar 

  43. Toma AI, Fuller JM, Willett NJ, Goudy SL. Oral wound healing models and emerging regenerative therapies. Transl Res. 2021;236:17–34. https://doi.org/10.1016/j.trsl.2021.06.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kong HK, Chan Z, Yan SW, et al. Revealing the species-specific genotype of the edible bird’s nest-producing swiftlet, Aerodramus fuciphagus and the proteome of edible bird’s nest. Food Res Int. 2022;160: 111670. https://doi.org/10.1016/j.foodres.2022.111670.

    Article  CAS  PubMed  Google Scholar 

  45. Xiao L, Wang DL. Nutrient composition and active components of edible bird nest. New Food Indust. 2022;64:579–82.

    Google Scholar 

  46. Wong ZCF, Chan GKL, Wu KQY, et al. Complete digestion of edible bird’s nest releases free N-acetylneuraminic acid and small peptides: an efficient method to improve functional properties. Food Funct. 2018;9:5139–49. https://doi.org/10.1039/c8fo00991k.

    Article  CAS  PubMed  Google Scholar 

  47. Lin YT, Shi QQ, Zhang L, et al. Hydrogen-rich water ameliorates neuropathological impairments in a mouse model of Alzheimer’s disease through reducing neuroinflammation and modulating intestinal microbiota. Neural Regen Res. 2022;17:409–17. https://doi.org/10.4103/1673-5374.317992.

    Article  CAS  PubMed  Google Scholar 

  48. Dhillon G, Buddhavarapu V, Grewal H, et al. Hydrogen water: extra healthy or a hoax? A systematic review. Int J Mol Sci. 2024;25:973. https://doi.org/10.3390/ijms25020973.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Saitoh Y, Harata Y, Mizuhashi F, Nakajima M, Miwa N. Biological safety of neutral-pH hydrogen-enriched electrolyzed water upon mutagenicity, genotoxicity and subchronic oral toxicity. Toxicol Ind Health. 2010;26:203–16. https://doi.org/10.1177/0748233710362989.

    Article  CAS  PubMed  Google Scholar 

  50. Chok KC, Ng MG, Ng KY, Koh RY, Tiong YL, Chye SM. Edible bird’s nest: recent updates and industry insights based on laboratory findings. Front Pharmacol. 2021;12: 746656. https://doi.org/10.3389/fphar.2021.746656.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was funded by JSPS KAKENHI Grant-in-Aid for Scientific Research (No. 22K10009 to L.X.; No. 21K21025 to N.S.). The administrative support and advice from Chikako Saiki, as well as Katsuhisa Sunada, are sincerely appreciated by the authors. Technical support from staffs in the Research Center for Odontology, School of Life Dentistry at Tokyo, The Nippon Dental University, are acknowledged by the authors. The authors also express gratitude to Nathaniel Green for his proofreading.

Funding

This research was funded by JSPS KAKENHI Grant-in-Aid for Scientific Research (No. 22K10009 to L.X.; No. 21K21025 to N.S.).

Author information

Author notes

  1. Dongliang Wang and Naohiro Shimamura have contributed equally to this work.

Authors and Affiliations

  1. Hebei Edible Bird’s Nest Fresh Stew Technology Innovation Center, Bazhou Economic Development Zone, Langfang, 065700, China

    Dongliang Wang

  2. Department of Dental Anesthesiology, School of Life Dentistry at Tokyo, The Nippon Dental University, Tokyo, Japan

    Naohiro Shimamura

  3. Prefectural University of Hiroshima, Faculty of Life Sciences, Hiroshima, 727-0023, Japan

    Nobuhiko Miwa

  4. Incorporated Association Hydrogen Medical Institute, Minatojima Minamicho 1-6-4, ChuOh-Ku, Kobe, 650-0047, Japan

    Nobuhiko Miwa

  5. Department of Physiology, School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-Ku, Tokyo, 102-8159, Japan

    Li Xiao

Authors
  1. Dongliang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naohiro Shimamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nobuhiko Miwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li Xiao.

Ethics declarations

Conflicts of interest

D.W. is an employee of Beijing Xiaoxiandun Biotechnology Co., Ltd. No other author has reported a potential conflict of interest relevant to this article.

Ethics approval

This study was approved by the Committee of Ethics at the Nippon Dental University School of Life Dentistry in Tokyo (authorization number: NDU-T2019-01).

Informed consent

Informed consent was obtained from all subjects involved in the study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, D., Shimamura, N., Miwa, N. et al. Combined use of hydrogen-rich water and enzyme-digested edible bird’s nest improves PMA/LPS-impaired wound healing in human inflammatory gingival tissue equivalents. Human Cell (2024). https://doi.org/10.1007/s13577-024-01065-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13577-024-01065-y

Keywords

Search

Navigation