Download PDF
CC BY-NC-ND 4.0 · Eur J Dent 2021; 15(02): 332-339
DOI: 10.1055/s-0040-1719213
Original Article

Gingival-Derived Mesenchymal Stem Cell from Rabbit (Oryctolagus cuniculus): Isolation, Culture, and Characterization

Alexander Patera Nugraha
1   Department of Medical Science, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
2   Department of Orthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
,
Fedik Abdul Rantam
3   Laboratory of Virology and Immunology, Department of Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia
,
Ida Bagus Narmada
2   Department of Orthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
,
Diah Savitri Ernawati
4   Department of Oral Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
,
Igo Syaiful Ihsan
5   Stem Cell Research and Development Center, Universitas Airlangga, Surabaya, Indonesia
› Author Affiliations
› Further Information
   Permissions and Reprints

Abstract

Objective This study aims to confirm whether the GDMSCs isolated from rabbit’s (Oryctolagus cuniculus) gingiva are mesenchymal stem cells (MSCs).

Materials and Methods This study design was partly quasi-experimental with an observational design. GDMSCs were isolated from the gingiva of healthy male rabbits (O. cuniculus) (n = 2), 6 months old, and 3 to 5 kg of body weight. The specific cell surface markers of MSCs; clusters of differentiation (CD), namely, CD44, CD73, CD90, CD105, and CD200 expressions; and hematopoietic stem cell surface markers CD34 and CD45 were examined using flow cytometry and immunohistochemistry with immunofluorescence. The osteogenic differentiation of isolated GDMSCs was examined using alizarin red staining.

Results GDMSCs in the fourth passage showed a spindle-like formation and fibroblast-like cells that attached to the base of the culture plate. GDMSCs were MSCs that positively expressed CD44, CD73, CD90, CD105, and CD200 but did not express CD34 and CD45 when examined using flow cytometry and immunohistochemical analysis. GDMSCs had osteogenic differentiation confirmed by calcified deposits in vitro with a red–violet and brownish color after alizarin red staining.

Conclusion GDMSCs isolated from the rabbits (O. cuniculus) were confirmed as MSCs in vitro documented using immunohistochemistry and flow cytometry. GDMSCs can differentiate into osteogenic lineage in vitro that may be suitable for regenerative dentistry.

Keywords

gingival-derived mesenchymal stem cells - cluster of differentiation - osteogenic differentiation - regenerative dentistry - tissue engineering


Publication History

Article published online:
01 December 2020

© 2020. European Journal of Dentistry. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, Second Floor, Sector -2, NOIDA -201301, India

 

  • References

  • 1 Nadig RR. Stem cell therapy - hype or hope? A review. J Conserv Dent 2009; 12 (04) 131-138
    CrossrefPubMedGoogle Scholar
  • 2 Saeedi P, Halabian R, Imani Fooladi AA. A revealing review of mesenchymal stem cells therapy, clinical perspectives and modification strategies. Stem Cell Investig 2019; 6: 34
    CrossrefPubMedGoogle Scholar
  • 3 Nugraha AP, Purwati. Susilowati H. et al. Medicinal signaling cells metabolite oral based as a potential biocompatible biomaterial accelerating oral ulcer healing (in vitro study). Eur Dent J 2019; 13 (03) 432-436
    Article in Thieme ConnectPubMedGoogle Scholar
  • 4 Prasetyo EP, Widjiastuti I, Cahyani F. et al. Cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells. Pesqui Bras Odontopediatria Clin Integr 2020; 20: 1-6
    CrossrefGoogle Scholar
  • 5 Kuntjoro M, Prasetyo EP, Cahyani F. et al. Lipopolysaccharide’s cytotoxicity on human umbilical cord mesenchymal stem cells. Pesqui Bras Odontopediatria Clin Integr 2020; 20 (e0048) 1-6
    CrossrefGoogle Scholar
  • 6 Rantam FA, Nugraha AP, Ferdiansyah F. et al. A potential differentiation of adipose and hair follicle-derived mesenchymal stem cells to generate neurons induced with EGF, FGF. Research J Pharm Tech 2020; 13 (01) 275-281
    CrossrefGoogle Scholar
  • 7 Sari DS, Maduratna E, Latief FDE, Nugraha AP, Sudiana K, Rantam FA. Ferdiansyah, Satuman. Osteogenic differentiation and biocompatibility of bovine teeth scaffold with rat adipose-derived mesenchymal stem cells. Eur J Dent 2019; 13 (02) 206-212
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Nugraha AP, Prasetyo EP, Kuntjoro M. et al. The effect of cobalt (II) chloride in the viability percentage and the induced hypoxia inducible factor -1αof human adipose mesenchymal stem cells (HAMSCs): an in vitro study. Sys Rev Pharm 2020; 11 (06) 308-314
    Google Scholar
  • 9 Narmada IB, Laksono V, Nugraha AP. et al. Regeneration of salivary gland defects of diabetic Wistar rats post human dental pulp stem cells intraglandular transplantation on acinar cell vacuolization and interleukin-10 serum level. Pesqui Bras Odontopediatria Clin Integr 2019; 19 (e5002) 1-10
    CrossrefGoogle Scholar
  • 10 Suciadi SP, Nugraha AP, Ernawati DS. et al. The efficacy of human dental pulp stem cells in regenerating submandibular gland defects in diabetic Wistar rats (Rattus novergicus). Res J Pharm Tech 2019; 12 (04) 1573-1579
    CrossrefGoogle Scholar
  • 11 Prahasanti C, Nugraha AP, Saskianti T, Suardita K, Riawan W, Ernawati DS. Exfoliated human deciduous tooth stem cells incorporating carbonate apatite scaffold enhance BMP-2, BMP-7 and attenuate MMP-8 expression during initial alveolar bone remodeling in Wistar rats (Rattus norvegicus). Clin Cosmet Investig Dent 2020; 12: 79-85
    CrossrefPubMedGoogle Scholar
  • 12 Sarasati A, Rezkita F, Wibawa KGP. et al. Gingival mesenchymal stem cells, concentrated growth factors and silk-fibroin scaffold to alleviate peripheral nerve regeneration: a review. Biochem Cell Arch 2020; 20 (Suppl. 01) 2921-2929
    Google Scholar
  • 13 Aprilianti NA, Rahmadhani D, Rizqianti Y, Ridwan RD, Ramadhani NF, Nugraha AP. Periodontal ligament stem cells, Solcoseryl pasta incorporated nano-hydroxyapatite silica gel scaffold for bone defect regeneration in chronic periodontitis: a review. Biochem Cell Arch 2020; 20 (Suppl. 01) 3101-3106
    Google Scholar
  • 14 Jin SH, Lee JE, Yun JH, Kim I, Ko Y, Park JB. Isolation and characterization of human mesenchymal stem cells from gingival connective tissue. J Periodontal Res 2015; 50 (04) 461-467
    CrossrefPubMedGoogle Scholar
  • 15 Du L, Yang P, Ge S. Isolation and characterization of human gingiva-derived mesenchymal stem cells using limiting dilution method. J Dent Sci 2016; 11 (03) 304-314
    CrossrefPubMedGoogle Scholar
  • 16 Fawzy El-Sayed KM, Dörfer CE. Gingival mesenchymal stem/progenitor cells: a unique tissue engineering gem. Stem Cells Int 2016; 2016: 7154327
    PubMedGoogle Scholar
  • 17 Sun Q, Nakata H, Yamamoto M, Kasugai S, Kuroda S. Comparison of gingiva-derived and bone marrow mesenchymal stem cells for osteogenesis. J Cell Mol Med 2019; 23 (11) 7592-7601
    CrossrefPubMedGoogle Scholar
  • 18 Sitasari PI, Narmada IB, Hamid T, Triwardhani A, Nugraha AP, Rahmawati D. East Java green tea methanolic extract can enhance RUNX2 and osterix expression during orthodontic tooth movement in vivo. J Pharm Pharmacogn Res 2020; 8 (04) 290-298
    Google Scholar
  • 19 Nugraha AP, Narmada IB, Ernawati DS. et al. Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio (in vitro). F1000 Res 2018; 7: 1134
    CrossrefPubMedGoogle Scholar
  • 20 Nugraha AP, Narmada IB, Ernawati DS. et al. In vitro bone sialoprotein-I expression in combined gingival stromal progenitor cells and platelet rich fibrin during osteogenic differentiation. Trop J Pharm Res 2018; 17 (12) 2341-2345
    CrossrefGoogle Scholar
  • 21 Nugraha AP, Narmada IB, Ernawati DS. et al. Bone alkaline phosphatase and osteocalcin expression of rat’s gingival mesenchymal stem cells cultured in platelet-rich fibrin for bone remodeling (in vitro study). Eur J Dent 2018; 12 (04) 566-573
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Nugraha AP, Narmada IB, Ernawatı DS. et al. The aggrecan expression post platelet rich fibrin administration in gingival medicinal signaling cells in Wistar rats (Rattus norvegicus) during the early osteogenic differentiation (in vitro). Kafkas Univ Vet Fak Derg 2019; 25 (03) 421-425
    Google Scholar
  • 23 Nugraha AP, Narmada IB, Ernawati DS. et al. Somatic cells acceleration by platelet rich fibrin. Indian Vet J 2019; 96 (04) 30-34
    Google Scholar
  • 24 Monterubbianesi R, Bencun M, Pagella P, Woloszyk A, Orsini G, Mitsiadis TA. A comparative in vitro study of the osteogenic and adipogenic potential of human dental pulp stem cells, gingival fibroblasts and foreskin fibroblasts. Sci Rep 2019; 9 (01) 1761
    CrossrefPubMedGoogle Scholar
  • 25 Vidoni C, Ferraresi A, Secomandi E. et al. Autophagy drives osteogenic differentiation of human gingival mesenchymal stem cells. Cell Commun Signal 2019; 17 (01) 98
    CrossrefPubMedGoogle Scholar
  • 26 Todd JL, Palmer SM. Danger signals in regulating the immune response to solid organ transplantation. J Clin Invest 2017; 127 (07) 2464-2472
    CrossrefPubMedGoogle Scholar
  • 27 Xia L, Peng R, Leng W. et al. TRAIL-expressing gingival-derived mesenchymal stem cells inhibit tumorigenesis of tongue squamous cell carcinoma. J Dent Res 2015; 94 (01) 219-228
    CrossrefPubMedGoogle Scholar
  • 28 Nugraha AP, Narmada IB, Ernawati DS. et al. Gingival mesenchymal stem cells from Wistar rat’s gingiva (Rattus norvegicus) – isolation and characterization (in vitro study). J Int Dent Med Res 2018; 11 (02) 694-699
    Google Scholar
  • 29 Cristaldi M, Mauceri R, Campisi G. et al. Growth and osteogenic differentiation of discarded gingiva-derived mesenchymal stem cells on a commercial scaffold. Front Cell Dev Biol 2020; 8: 292
    CrossrefPubMedGoogle Scholar
  • 30 Dominici M, Le Blanc K, Mueller I. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8 (04) 315-317
    CrossrefPubMedGoogle Scholar
  • 31 L. Ramos T, Sánchez-Abarca LI, Muntión S. et al. MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. Cell Commun Signal 2016; 14: 2
    CrossrefPubMedGoogle Scholar
  • 32 Monguió-Tortajada M, Roura S, Gálvez-Montón C, Franquesa M, Bayes-Genis A, Borràs FE. Mesenchymal stem cells induce expression of CD73 in human monocytes in vitro and in a swine model of myocardial infarction in vivo. . Front Immunol 2017; 8: 1577
    CrossrefPubMedGoogle Scholar
  • 33 Breitbach M, Kimura K, Luis TC. et al. In vivo labeling by CD73 marks multipotent stromal cells and highlights endothelial heterogeneity in the bone marrow niche. Cell Stem Cell 2018; 22 (02) 262-276.e7
    CrossrefPubMedGoogle Scholar
  • 34 Moraes DA, Sibov TT, Pavon LF. et al. A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells. Stem Cell Res Ther 2016; 7 (01) 97
    CrossrefPubMedGoogle Scholar
  • 35 Nassiri F, Cusimano MD, Scheithauer BW. et al. Endoglin (CD105): a review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Res 2011; 31 (06) 2283-2290
    Google Scholar
  • 36 Levi B, Wan DC, Glotzbach JP. et al. CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor β1 (TGF-β1) signaling. J Biol Chem 2011; 286 (45) 39497-39509
    CrossrefPubMedGoogle Scholar
  • 37 Minas K, Liversidge J. Is the CD200/CD200 receptor interaction more than just a myeloid cell inhibitory signal. ? Crit Rev Immunol 2006; 26 (03) 213-230
    CrossrefPubMedGoogle Scholar
  • 38 Pontikoglou C, Langonné A, Ba MA. et al. CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro-osteogenic and pro-inflammatory cues. J Cell Mol Med 2016; 20 (04) 655-665
    CrossrefPubMedGoogle Scholar
  • 39 AbuSamra DB, Aleisa FA, Al-Amoodi AS. et al. Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44. Blood Adv 2017; 1 (27) 2799-2816
    CrossrefPubMedGoogle Scholar
  • 40 Altin JG, Sloan EK. The role of CD45 and CD45-associated molecules in T cell activation. Immunol Cell Biol 1997; 75 (05) 430-445
    CrossrefPubMedGoogle Scholar
  • 41 Pavone V, Testa G, Giardina SMC, Vescio A, Restivo DA, Sessa G. Pharmacological therapy of osteoporosis: a systematic current review of literature. Front Pharmacol 2017; 8 (11) 803
    CrossrefPubMedGoogle Scholar
  • 42 Tomasevic-Todorovic S, Vazic A, Issaka A, Hanna F. Comparative assessment of fracture risk among osteoporosis and osteopenia patients: a cross-sectional study. Open Access Rheumatol 2018; 10: 61-66
    CrossrefPubMedGoogle Scholar
  • 43 Angelopoulos I, Brizuela C, Khoury M. Gingival mesenchymal stem cells outperform haploidentical dental pulp-derived mesenchymal stem cells in proliferation rate, migration ability, and angiogenic potential. Cell Transplant 2018; 27 (06) 967-978
    CrossrefPubMedGoogle Scholar
  • 44 Xu QC, Wang ZG, Ji QX. et al. Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration. Int J Clin Exp Pathol 2014; 7 (08) 4922-4929
    PubMedGoogle Scholar
  • 45 Liu H, Li M, Du L, Yang P, Ge S. Local administration of stromal cell-derived factor-1 promotes stem cell recruitment and bone regeneration in a rat periodontal bone defect model. Mater Sci Eng C 2015; 53: 83-94
    CrossrefPubMedGoogle Scholar
  • 46 Fawzy-El-Sayed K, Mekhemar M, Adam-Klages S, Kabelitz D, Dörfer C. TlR expression profile of human gingival margin-derived stem progenitor cells. Med Oral Patol Oral Cir Bucal 2016; 21 (01) e30-e38
    CrossrefPubMedGoogle Scholar
  • 47 Alhasyimi AA, Rosyida NF, Rihadini MS. Postorthodontic relapse prevention by administration of grape seed (Vitis vinifera) extract containing cyanidine in rats. Eur J Dent 2019; 13 (04) 629-634
    Article in Thieme ConnectPubMedGoogle Scholar
  • 48 Alhasyimi AA, Pudyani PP, Asmara W, Ana ID. Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits. Orthod Craniofac Res 2018; 21 (02) 112-118
    CrossrefPubMedGoogle Scholar