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2017, Number 1

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Rev Odont Mex 2017; 21 (1)

Isolation of periodontal ligament stem cells from extracted premolars. Simplified method

Trejo ICG, Ramírez RO, Muñoz GA, Verdín TSL, Gómez CJF

Language: Spanish
References: 22
Page: 13-21
PDF size: 494.30 Kb.


ABSTRACT

Periodontal ligament is a fibrous connective tissue of ectomesenchymal origin which contains a multipotent postnatal stem cells population with clonogenic capacity and high proliferation index. Multipotent postnatal stem cells population culture has been achieved through several extraction methods, nevertheless some technical problems have been encountered. For this reason, in the present study, 6 different methods were compared so as to improve isolation of primary periodontal ligament cells. Multipotent postnatal stem cells population were isolated from 36 healthy premolars, which were then cultured in complete Dulbecco modified Eagle medium (BioWest) (added with L-glutamine, 10% fetal bovine serum and 1% antibiotic), in standard culture conditions. Assays for cell proliferation were conducted with 3-(4,5 dimethylthiazol-2yl)-2,5 diphenyltetrazolium technique as well as cell migration through crop injury test, after which osteogenic differentiation was assessed. Cultures were performed in triplicate with each of the published techniques; thus we were able to obtain a minimum population of cells susceptible to contamination. The «simplified method» was conducted through explant enzymatic digestion and culture: with obtained multipotent postnatal stem cells population, a 14 day proliferation was observed in standard conditions of ascendant culture; in migration tests, multipotent postnatal stem cells population totally covered injured surfaces after 72 hours. CD90 positive expression was observed in over 80% of all cells; CD73 positive expression was found in 70% of cells, CD105 in 60% and vimentin in 90% of the population, as well as negative marking to CD11b and CD45. Positive osteogenic differentiation was observed after 14 days, through positive expression to RUNX2 in at least 15% of cells, and to osteocalcin in 90% as well as positive test to alizarin red. Based on obtained results we were able to affirm that the simplified method allows to isolate a population of mesenchymal cells, which might be obtained from extracted healthy premolars.


REFERENCES

  1. Xiao L, Nasu M. From regenerative dentistry to regenerative medicine: progress, challenges, and potential applications of oral stem cells. Stem Cells Cloning. 2014; 7: 89-99.

  2. Ivanovski S, Gronthos S, Shi S, Bartold PM. Stem cells in the periodontal ligament. Oral Dis. 2006; 12 (4): 358-363.

  3. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004; 364 (9429): 149-155.

  4. Seymour GJ, Cullinan MP, Heng NC. Oral biology. Molecular techniques and applications. New York: Springer; 2010.

  5. Zhu W, Liang M. Periodontal ligament stem cells: current status, concerns, and future prospects. Stem Cells Int. 2015; 2015: 972313.

  6. Navabazam AR, Sadeghian-Nodoshan F, Sheikhha MH, Miresmaeili SM, Soleimani M, Fesahat F. Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament. Iran J Reprod Med. 2013; 11 (3): 235-242.

  7. Trubiani O, Di Primio R, Traini T, Pizzicannella J, Scarano A, Piattelli A et al. Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament. Int J Immunopathol Pharmacol. 2005; 18 (2): 213-221.

  8. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S et al. Stem cell properties of human periodontal ligament cells. J Periodontal Res. 2006; 41 (4): 303-310.

  9. Saito MT, Silverio KG, Casati MZ, Sallum EA, Nociti FH Jr. Tooth-derived stem cells: update and perspectives. World J Stem Cells. 2015; 7 (2): 399-407.

  10. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res. 2005; 84 (10): 907-912.

  11. Guo S, Guo W, Ding Y, Gong J, Zou Q, Xie D et al. Comparative study of human dental follicle cell sheets and periodontal ligament cell sheets for periodontal tissue regeneration. Cell Transplant. 2013; 22 (6): 1061-1073.

  12. Ding G, Liu Y, Wang W, Wei F, Liu D, Fan Z et al. Allogeneic periodontal ligament stem cell therapy for periodontitis in swine. Stem Cells. 2010; 28 (10): 1829-1838.

  13. Su F, Liu SS, Ma JL, Wang DS, E LL, Liu HC. Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells. Stem Cell Res Ther. 2015; 6: 22.

  14. Yang H, Gao LN, An Y, Hu CH, Jin F, Zhou J et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions. Biomaterials. 2013; 34 (29): 7033-7047.

  15. Xu Q, Li B, Yuan L, Dong Z, Zhang H, Wang H et al. Combination of platelet-rich plasma within periodontal ligament stem cell sheets enhances cell differentiation and matrix production. J Tissue Eng Regen Med. 2014. doi: 10.1002/term.1953.

  16. Akizuki T, Oda S, Komaki M, Tsuchioka H, Kawakatsu N, Kikuchi A et al. Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs. J Periodont Res. 2005; 40 (3): 245-251.

  17. Tran-Hle B, Doan VN, Le HT, Ngo LT. Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine. In vitro Cell Dev Biol Anim. 2014; 50 (7): 597-602.

  18. Paladino FV, Peixoto-Cruz JS, Santacruz-Perez C, Goldberg AC. Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells. Cell Tissue Bank. 2016; 17 (1): 123-136.

  19. Karamzadeh R, Eslaminejad MB, Aflatoonian R. Isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods. J Vis Exp. 2012; (69). pii: 4372. doi: 10.3791/4372.

  20. Domenis R, Lazzaro L, Calabrese S, Mangoni D, Gallelli A, Bourkoula E et al. Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques. Stem Cell Res Ther. 2015; 6: 2.

  21. Iwasaki K, Komaki M, Yokoyama N, Tanaka Y, Taki A, Honda I et al. Periodontal regeneration using periodontal ligament stem cell-transferred amnion. Tissue Eng Part A. 2014; 20 (3-4): 693-704.

  22. Cha Y, Jeon M, Lee HS, Kim S, Kim SO, Lee JH et al. Effects of in vitro osteogenic induction on in vivo tissue regeneration by dental pulp and periodontal ligament stem cells. J Endod. 2015; 41 (9): 1462-1468.




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