Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells F. J. Rodrı ´guez-Lozano • D. Garcı ´a-Bernal • S. Aznar-Cervantes • M. A. Ros-Roca • M. C. Alguero ´ • N. M. Atucha • A. A. Lozano-Garcı ´a • J. M. Moraleda • J. L. Cenis Received: 9 March 2014 / Accepted: 21 July 2014 / Published online: 1 August 2014 Ó Springer Science+Business Media New York 2014 Abstract In regenerative dentistry, stem cell-based ther- apy 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 engi- neering as they are both non toxic and promote cell pro- liferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The pur- pose of this study was to investigate the effects of com- posite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and pro- liferation 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 pro- liferation. 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. 1 Introduction Graphene is a recent addition to the family of carbon-based materials such as graphite, fullerenes, nanotubes, nano- wires and nanoribbons. It is a sheet of two-dimensional single layer sp 2 hybridized carbon atoms in a honeycomb lattice configuration [1]. Because of the singularity of its molecular configuration, graphene shows outstanding properties. It has a high electric and thermal conductivities, and high mechanical strength, as well as excellent optical properties. Thus, graphene is being developed in a con- siderable diversity of applications in fields as diverse as energy conversion [2] biosensing [3], imaging [4] and the F. J. Rodrı ´guez-Lozano (&) Á D. Garcı ´a-Bernal Á M. A. Ros-Roca Á M. C. Alguero ´ Á N. M. Atucha Á J. M. Moraleda Hematopoietic Transplant and Cellular Therapy Unit, Hematology Department, Virgen de la Arrixaca Clinical University Hospital, IMIB-Arrixaca, University of Murcia, Murcia, Spain e-mail: fcojavier@um.es F. J. Rodrı ´guez-Lozano School of Dentistry, Faculty of Medicine, University of Murcia, Murcia, Spain S. Aznar-Cervantes Á A. A. Lozano-Garcı ´a Á J. L. Cenis Department of Biotechnology, Instituto Murciano de Investigacio ´n y Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain N. M. Atucha Department of Physiology, Faculty of Medicine, University of Murcia, Murcia, Spain 123 J Mater Sci: Mater Med (2014) 25:2731–2741 DOI 10.1007/s10856-014-5293-2