Protective Effects of Membrane-Free Stem Cell Extract from H2O2-Induced Inflammation Responses in Human Periodontal Ligament Fibroblasts

무막줄기세포추출물의 H2O2에 의해 유도된 치주 세포의 염증 반응 보호 효과

  • He, Mei Tong (Department of Food Science and Nutrition, Pusan National University) ;
  • Kim, Ji Hyun (Department of Food Science and Nutrition, Pusan National University) ;
  • Kim, Young Sil (T-STEM Co., Ltd.) ;
  • Park, Hye Sook (T-STEM Co., Ltd.) ;
  • Cho, Eun Ju (Department of Food Science and Nutrition, Pusan National University)
  • Received : 2019.02.26
  • Accepted : 2019.06.07
  • Published : 2019.06.30


Periodontal inflammation, a major kind of periodontal diseases, is characterized to bleed, pain, and teeth loss, and it is resulted from oxidative stress. Membrane-free stem cell extract could avoid the immunogencity rejection by removal of cell membrane. In the present study, we investigated the protective effect of membrane-free stem cell extract from oxidative stress-induced periodontal inflammation in human periodontal ligament fibroblasts (HPLF). In the cell viability measurement, membrane-free stem cell extract showed significant increase of cell viability, compared with the $H_2O_2$-treated control group. To further investigation of molecular mechanisms, we measured inflammation and apoptosis related protein expressions. Membrane-free stem cell extract attenuated inflammation-related protein expressions such as nuclear factor kappa light chain enhancer of activated B cells, inducible nitric oxide synthase, and interleukin-6. In addition, the treatment of membrane-free stem cell extract decreased apoptotic protein expressions such as cleaved caspase-9, -3, poly (ADP-ribose) polymerase, and B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 ratio in the $H_2O_2$-treated HPLF cells. In conclusion, membrane-free stem cell extract exhibited anti-oxidative stress effects by regulation of inflammation and apoptosis in HPLF, suggesting that it could be used as the treatment agents for periodontal inflammatory disease.

SHGSCZ_2019_v20n6_95_f0001.png 이미지

Fig. 1. Effect of membrane-free stem cell extract on H2O2-treated HPLF cells. Values are presented as mean ± SD. The different letters (a-c) indicate significant differences (P < 0.05) by Duncan’s multiple range test.

SHGSCZ_2019_v20n6_95_f0002.png 이미지

Fig. 2. Effect of membrane-free stem cell extract on inflammation-related protein expression in H2O2-treated HPLF cells. Values are presented as mean ± SD. The different letters (a-e) indicate significant differences (P < 0.05) by Duncan’s multiple range test.

SHGSCZ_2019_v20n6_95_f0003.png 이미지

Fig. 3. Effect of membrane-free stem cell extract on apoptosis-related protein expression such as caspase-9, -3, and PARP in H2O2-treated HPLF cells. Values are presented as mean ± SD. The different letters (a-d) indicate significant differences (P < 0.05) by Duncan’s multiple range test.

SHGSCZ_2019_v20n6_95_f0004.png 이미지

Fig. 4. Effect of membrane-free stem cell extract on apoptosis-related protein expression such as Bax and Bcl-2 in H2O2-treated HPLF cells. Values are presented as mean ± SD. The different letters (a-d) indicate significant differences (P < 0.05) by Duncan’s multiple range test.

Table 1. Cytotoxicity of membrane-free stem cell extract on HPLF cells

SHGSCZ_2019_v20n6_95_t0001.png 이미지


  1. B. L. Pihlstrom, B. S. Michalowicz, N. W. Johnson, "Periodontal diseases", The Lancet, Vol.366, No.9499, pp.1809-1820, 2005. DOI:
  2. Y. H. Kim, B. S. Park, "Antioxidant effect of eugenol in human periodontal ligament fibroblasts", Korean Journal of Physical Anthropology, Vol.28, No.1, pp.45-53, 2015. DOI:
  3. J. E. Seo, E. S. Hwang, G. H. Kim, "Antioxidative and differentiation effects of Artemisia capillaris T. extract on hydrogen peroxide-induced oxidative damage of MC3T3-E1 osteoblast cells", Journal of the Korean Society of Food Science and Nutrition, Vol.40, No.11, pp.1532-1536, 2011. DOI:
  4. Y. Choe, J. Y. Yu, Y. O. Son, S. M. Park, J. G. Kim, X. Shi, J. C. Lee, "Continuously generated $H_2O_2$ stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts", Journal of Cellular Biochemistry, Vol.113, No.4, pp.1426-1423, 2012. DOI:
  5. C. F. Canakci, Y. Cicek, V. Canakci, "Reactive oxygen species and human inflammatory periodontal diseases", Biochemistry (Moscow), Vol.70, No.6, pp.619-628, 2005. DOI:
  6. N. S. Hwang, C. Zhang, Y. S. Hwang, S. Varghese, "Mesenchymal stem cell differentiation and roles in regenerative medicine", Systems Biology and Medicine, Vol.1, No.1, pp.97-106, 2009. DOI:
  7. A. Goldberg, K. Mitchell, J. Soans, L. Kim, R. Zaidi. "The use of mesenchymal stem cells for carilage repair and regeneration: a systematic review", Journal of Orthopaedic Surgery and Research, Vol.12, No.1, pp.39, 2017. DOI:
  8. J. W. Rhie, K. J. Kim, "Adipose stem cell therapy: present, future", Journal of Korean Wound Management Society, Vol.12, No.2, pp.39-45, 2016.
  9. S. Kern, H. Eichler, J. Stoeve, H. Kluter, K. Bieback, "Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue", Stem Cells, Vol.24, No.5, pp.1294-1301, 2006. DOI:
  10. H. A. Makhluf, S. M. Mueller, S. Mizuno, J. Glowacki, "Age-related decline in osteoprotegerin expression by human bone marrow cells cultured in three-dimensional collagen sponges", Biochemical and Biophysical Research Communications, Vol.268, No.3, pp.669-672, 2000. DOI:
  11. Y. Zhu, T. Liu, K. Song, X. Fan, X. Ma, Z. Cui, "Adipose-derived stem cell: a better stem cell than BMSC", Cell Biochemistry and Function, Vol.26, No.6, pp.664-675, 2008. DOI:
  12. M. Tobita, H. Mizuno. "Adipose-derived stem cells for periodontal tissue regeneration.", Methods in Molecular Biology, Vol.702, pp.461-470, 2011. DOI:
  13. F. J. Vizoso, N. Eiro, S. Cid, J. Schneider, R. Perez-Fernandez, "Mesenchymal stem cell secretome: toward cell-free therapeutic strategies in regenerative medicine." International Journal of Molecular Sciences, Vol.18, No.9, pp.E1852, 2017. DOI:
  14. Y. S. Kim, J. T. Lim, "Golf injury therapy using stem cell", Journal of Golf Studies, Vol.11, No.3, pp.55-65, 2017.
  15. E. Mohammed, E. Khalil, D. Sabry. "Effect of adipose-derived stem cells and their exo as adjunctive therapy to nonsurgical periodontal treatment: a histologic and histomorphometric study in rats." Biomolecules, Vol.8, No.4, pp.E167, 2018. DOI:
  16. T. Mosmann, "Rapid colormetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.", Journal of Immunological Methods, Vol.65, pp.55-63, 1983.
  17. C. Wittmann, P. Chockley, S. K. Singh, L. Pase, G. J. Lieschke, C. Grabher, "Hydrogen peroxide in inflammation: messenger, guide, and assassin", Advances in Hematology, Vol.2012, 2012. DOI:
  18. Y. Yamamoto, R. B. Gaynor, "Role of the NF-kB pathway in the pathogenesis of human disease states", Current Molecular Medicine, Vol.1, No.3, pp.287-296, 2001. DOI:
  19. C. S. Kim, T. Kawada, B. S. Kim, I. S. Han, S. Y. Choe, T. Kurata, R. Yu, "Capsaicin exhibits anti-inflammatory property by inhibiting IkB-a degradation in LPS-stimulated peritoneal macrophages", Cellular Signalling, Vol.15, No.3, pp.299-306, 2003. DOI:
  20. S. M. Park, R. J. Zhao, J. R. Lee, C. W. Lee, H. J. Kim, Y. K. Kwon, S. C. Kim, "Inhibitory effect of oyster conchioloin on pro-inflammatory mediator in lipopolysaccharide-activated Raw 264.7 cells", Journal of Physiology & Pathology in Korean Medicine, Vol.22, No.4, pp.878-883, 2008.
  21. S. I. Lee, J. K. Yi, W. J. Bae, S. Lee, H. J. Cha, E. C. Kim, "Thymosin beta-4 suppresses osteoclastic differentiation and inflammatory responses in human periodontal ligament cells", Plos One, Vol.11, No.1, pp.e0146708, 2016. DOI:
  22. L. Golz, S. Memmert, B. Rath-Deschner, A. Jager, T. Appel, G. Baumgarten, W. Gotz, S. Frede, "LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis", Mediators of Inflammation, Vol.2014, No.2014, pp.986264, 2014. DOI:
  23. M. I. Guillen, J. Platas, P del. Caz, M. Dolores, V. Mirabet, M. J. Alcaraz, "Paracrine anti-inflammatory effects of adipose tissue-derived mesenchymal stem cells in human monocytes", Frontiers in Physiology, Vol.9, pp.661, 2018. DOI:
  24. Y. B. Chae, J. S. Lee, H. J. Park, I. H. Park, M. M. Kim, Y. H. Park, D. S. Kim, J. H. Lee, "Advanced adipose-derived stem cell protein extracts with antioxidant activity modulates matrix metalloproteinases in human dermal fibroblasts", Environmental Toxicology and Pharmacology, Vol.34, No.2, pp.263-271, 2012. DOI:
  25. S. Elmore, "Apoptosis: a review of programmed cell death", Toxicologic Pathology, Vol.35, No.4, pp.495-516, 2007. DOI:
  26. K. Kannan, S. K. Jain, "Oxidative stress and apoptosis", Pathophysiology, Vol.7, No.3, pp.153-613, 2000. DOI:
  27. C. Soldani, A. I. Scovassi, "Poly (ADP-ribose) polymerase-1 cleavage during apoptosis: an update", Apoptosis, Vol.7, No.4, pp.321-328, 2002. DOI:
  28. Y. J. Chiu, J. S. Yang, H. S. Hsu, C. H. Tsai, H. Ma, "Adipose-derived stem cell conditioned medium attenuates cisplatin-triggered apoptosis in tongue squamous cell carcinoma", Oncology Reports, Vol.1, No.39, pp.651-658, 2018.
  29. K. J. Botting, K. C. Wang, M. Padhee, I. C. McMillen, B. Summers-Pearce, L. Rattanatray, N. Cutri, G. S. Posterino, D. A. Brooks, J. L. Morrison, "Early origins of heart disease: low birth weight and determinants of cardiomyocyte endowment," Clinical and Experimental Pharmacology and Physiology, Vol.39, No.9, pp.814-823, 2012. DOI:
  30. T. Liu, M. Lee, J. J. Ban, W. Im, I. M. Jung, M, Kim, "Cytosolic extract of human adipose stem cells reverses the amyloid beta-induced mitochondrial apoptosis via P53/Foxo3a pathway", Plos One, Vol.3, No.12, e0168859, 2017. DOI:
  31. A. Bergmann, H. Steller, "Apoptosis, stem cells, and tissue regeneration", Science Signaling, Vol.3, No.145, pp.8, 2010. DOI: