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Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells

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Abstract

In regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Graphene oxide (GO) and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The purpose of this study was to investigate the effects of composite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and proliferation rate of PDLSCs. PDLSCs obtained from healthy extracted teeth were cultured on GO, SF or combination of GO and SF films up to 10 days. Adhesion level of PDSCs on the different biomaterials were evaluated after 12 h of culture, whereas proliferation rate of cells was assessed using the MTT assay. Level of apoptosis was determined using Annexin-V and 7-AAD and mesenchymal markers expression of PDLSCs were analyzed by flow cytometry. At day 7 of culture, MTT experiments showed a high rate of proliferation of PDLSCs growing on GO films compared to the other tested biomaterials, although it was slightly lower than in plastic (control). However PDLSCs growing in fibroin or GO plus fibroin films showed a discrete proliferation. Importantly, at day 10 of culture it was observed a significant increase in PDLSCs proliferation rate in GO films compared to plastic (P < 0.05), as well as in GO plus fibroin compared to fibroin alone (P < 0.001). Flow cytometry analysis showed that culture of PDLSCs in fibroin, GO or GO plus fibroin films did not significantly alter the level of expression of the mesenchymal markers CD73, CD90 or CD105 up to 168 h, being the cell viability in GO even better than obtained in plastic. Our findings suggest that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs have strong potential for their therapeutic use in regenerative dentistry.

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References

  1. Allen MJ, Tung VC, Kaner RB. Honeycomb carbon: a review of graphene. Chem Rev. 2010;110(1):132–45.

    Article  Google Scholar 

  2. Sahoo NG, Pan Y, Li L, Chan SH. Graphene-based materials for energy conversion. Adv Mater. 2012;24(30):4203–10.

    Article  Google Scholar 

  3. Pumera M, Ambrosi A, Bonanni A, Chng ELK, Poh HL. Graphene for electrochemical sensing and biosensing. TrAC Trends Anal Chem. 2010;29(9):954–65.

    Article  Google Scholar 

  4. Yao J, Sun Y, Yang M, Duan Y. Chemistry, physics and biology of graphene-based nanomaterials: new horizons for sensing, imaging and medicine. J Mater Chem. 2012;22:14313–29.

    Article  Google Scholar 

  5. Geim AK. Graphene: status and prospects. Science. 2009;324(5934):1530–4.

    Article  Google Scholar 

  6. Fernández-Merino MJ, Guardia L, Paredes JI, Villar-Rodil S, Solís-Fernández P, et al. Vitamin C is an ideal substitute for hydrazine in the reduction of graphene oxide suspensions. J Phys Chem C. 2010;114:6426–32.

    Article  Google Scholar 

  7. Yang K, Li Y, Tan X, Peng R, Liu Z. Behavior and toxicity of graphene and its functionalized derivatives in biological systems. Small. 2013;9(9–10):1492–503.

    Article  Google Scholar 

  8. Chung C, Kim YK, Shin D, Ryoo SR, Hong BH, Min DH. Biomedical applications of graphene and graphene oxide. Acc Chem Res. 2013;46(10):2211–24.

    Article  Google Scholar 

  9. Shen H, Zhang L, Liu M, Zhang Z. Biomedical applications of graphene. Theranostics. 2012;2(3):283–94.

    Article  Google Scholar 

  10. Kalbacova M, Broz A, Kong J, Kalbac M. Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells. Carbon. 2010;48(15):4323–9.

    Article  Google Scholar 

  11. Li N, Zhang X, Song Q, Su R, Zhang Q, Kong T, et al. The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates. Biomaterials. 2011;32(35):9374–82.

    Article  Google Scholar 

  12. Akhavan O, Ghaderi E, Abouei E, Hatamie S, Ghasemi E. Accelerated differentiation of neural stem cells into neurons on ginseng-reduced graphene oxide sheets. Carbon. 2014;66:395–406.

    Article  Google Scholar 

  13. Nayak TR, Andersen H, Makam VS, Khaw C, Bae S, Xu X, et al. Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells. ACS Nano. 2011;5(6):4670–8.

    Article  Google Scholar 

  14. Park SY, Park J, Sim SH, Sung MG, Kim KS, Hong BH, et al. Enhanced differentiation of human neural stem cells into neurons on graphene. Adv Mater. 2011;23(36):H263–7.

    Article  Google Scholar 

  15. Chen GY, Pang DW, Hwang SM, Tuan HY, Hu YC. A graphene-based platform for induced pluripotent stem cells culture and differentiation. Biomaterials. 2012;33(2):418–27.

    Article  Google Scholar 

  16. Goenka S, Sant V, Sant S. Graphene-based nanomaterials for drug delivery and tissue engineering. J Control Release. 2014;173:75–88.

    Article  Google Scholar 

  17. Verdejo R, Bernal MM, Romasanta LJ, Lopez-Manchado MA. Graphene filled polymer nanocomposites. J Mater Chem. 2011;21:3301–10.

    Article  Google Scholar 

  18. Hu K, Gupta MK, Kulkarni DD, Tsukruk VV. Ultra-robust graphene oxide-silk fibroin nanocomposite membranes. Adv Mater. 2013;25(16):2301–7.

    Article  Google Scholar 

  19. Hu K, Tolentino LS, Kulkarni DD, Ye C, Kumar S, Tsukruk VV. Written-in conductive patterns on robust graphene oxide biopaper by electrochemical microstamping. Angew Chem Int Ed Engl. 2013;52(51):13784–8.

    Article  Google Scholar 

  20. Rodríguez-Lozano FJ, Insausti CL, Iniesta F, Blanquer M, Ramírez MD, Meseguer L, et al. Mesenchymal dental stem cells in regenerative dentistry. Med Oral Patol Oral Cir Bucal. 2012;17(6):e1062–7.

    Article  Google Scholar 

  21. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA. 2000;97:13625–30.

    Article  Google Scholar 

  22. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003;100:5807–12.

    Article  Google Scholar 

  23. 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;364:149–55.

    Article  Google Scholar 

  24. Morsczeck C, Götz W, Schierholz J, Zeilhofer F, Kühn U, Möhl C, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 2005;24:155–65.

    Article  Google Scholar 

  25. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One. 2006;1:e79.

    Article  Google Scholar 

  26. Rodríguez-Lozano FJ, Bueno C, Insausti CL, Meseguer L, Ramírez MC, Blanquer M, et al. Mesenchymal stem cells derived from dental tissues. Int Endod J. 2011;44:800–6.

    Article  Google Scholar 

  27. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7.

    Article  Google Scholar 

  28. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, et al. Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement. Cytotherapy. 2005;7:393–5.

    Article  Google Scholar 

  29. Etienne O, Schneider A, Kluge JA, Bellemin-Laponnaz C, Polidori C, Leisk GG, et al. Soft tissue augmentation using silk gels: an in vitro and in vivo study. J Periodontol. 2009;80(11):1852–8.

    Article  Google Scholar 

  30. Park J, Park S, Ryu S, Bhang SH, Kim J, Yoon JK, et al. Graphene-regulated cardiomyogenic differentiation process of mesenchymal stem cells by enhancing the expression of extracellular matrix proteins and cell signaling molecules. Adv Healthc Mater. 2014;3(2):176–81.

    Article  Google Scholar 

  31. Kim TH, Lee KB, Choi JW. 3D graphene oxide-encapsulated gold nanoparticles to detect neural stem cell differentiation. Biomaterials. 2013;34(34):8660–70.

    Article  Google Scholar 

  32. Bueno C, Ramirez C, Rodríguez-Lozano FJ, Tabarés-Seisdedos R, Rodenas M, Moraleda JM, et al. Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain. Cell Transpl. 2013;22(11):2017–28.

    Article  Google Scholar 

  33. Crowder SW, Prasai D, Rath R, Balikov DA, Bae H, Bolotin KI, et al. Three-dimensional graphene foams promote osteogenic differentiation of human mesenchymal stem cells. Nanoscale. 2013;5(10):4171–6.

    Article  Google Scholar 

  34. Li X, Liu H, Niu X, Yu B, Fan Y, Feng Q, et al. The use of carbon nanotubes to induce osteogenic differentiation of human adipose-derived MSCs in vitro and ectopic bone formation in vivo. Biomaterials. 2012;33(19):4818–27.

    Article  Google Scholar 

  35. Aryaei A, Jayatissa AH, Jayasuriya AC. The effect of graphene substrate on osteoblast cell adhesion and proliferation. J. Biomed Mater Res A. 2013. doi:10.1002/jbm.a.34993.

    Google Scholar 

  36. Chi NH, Yang MC, Chung TW, Chen JY, Chou NK, Wang SS. Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials. 2012;33(22):5541–51.

    Article  Google Scholar 

  37. Riccio M, Maraldi T, Pisciotta A, La Sala GB, Ferrari A, Bruzzesi G. Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells. Tissue Eng Part A. 2012;18(9–10):1006–13.

    Article  Google Scholar 

  38. Lawrence BD, Marchant JK, Pindrus MA, Omenetto FG, Kaplan DL. Silk film biomaterials for cornea tissue engineering. Biomaterials. 2009;30(7):1299–308.

    Article  Google Scholar 

  39. Madden PW, Lai JN, George KA, Giovenco T, Harkin DG, Chirila TV. Human corneal endothelial cell growth on a silk fibroin membrane. Biomaterials. 2011;32(17):4076–84.

    Article  Google Scholar 

  40. Rodríguez-Lozano FJ, Serrano-Belmonte I, Pérez Calvo JC, Coronado-Parra MT, Bernabeu-Esclapez A, Moraleda JM. Effects of two low-shrinkage composites on dental stem cells (viability, cell damaged or apoptosis and mesenchymal markers expression). J Mater Sci Mater Med. 2013;24(4):979–88.

    Article  Google Scholar 

  41. Akhavan O, Ghaderi E, Akhavan A. Size-dependent genotoxicity of graphene nanoplatelets in human stem cells. Biomaterials. 2012;33(32):8017–25.

    Article  Google Scholar 

  42. Liu TL, Miao JC, Sheng WH, Xie YF, Huang Q, Shan YB, et al. Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to wound healing. J Zhejiang Univ Sci B. 2010;11(1):10–6.

    Article  Google Scholar 

Download references

Acknowledgments

We thank the teeth donors for their generosity. This work was supported by FIS EC07/90762 Grant and the Spanish Net of Cell Therapy (TerCel) provided by Carlos III Institute of Health (ISCiii) (RETICS RD07/0010/2012 and RD12/0019/0001) together with the Junction Program for Biomedical Research in Advanced Therapies and Regenerative Medicine from ISCiii and FFIS, and by the Fundación Seneca (Grant 08859/PI/08). Experiments made at the IMIDA were supported by funds from UE FEDER Operative Program Region of Murcia 2007–2013.

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Authors and Affiliations

  1. Hematopoietic Transplant and Cellular Therapy Unit, Hematology Department, Virgen de la Arrixaca Clinical University Hospital, IMIB-Arrixaca, University of Murcia, Murcia, Spain

    F. J. Rodríguez-Lozano, D. García-Bernal, M. A. Ros-Roca, M. C. Algueró, N. M. Atucha & J. M. Moraleda

  2. School of Dentistry, Faculty of Medicine, University of Murcia, Murcia, Spain

    F. J. Rodríguez-Lozano

  3. Department of Physiology, Faculty of Medicine, University of Murcia, Murcia, Spain

    N. M. Atucha

  4. Department of Biotechnology, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain

    S. Aznar-Cervantes, A. A. Lozano-García & J. L. Cenis

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  1. F. J. Rodríguez-Lozano
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  2. D. García-Bernal
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Correspondence to F. J. Rodríguez-Lozano.

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Rodríguez-Lozano, F.J., García-Bernal, D., Aznar-Cervantes, S. et al. Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells. J Mater Sci: Mater Med 25, 2731–2741 (2014). https://doi.org/10.1007/s10856-014-5293-2

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