Covalent Decoration of Cortical Membranes with Graphene Oxide as a Substrate for Dental Pulp Stem Cells

Roberta Di Carlo, Susi Zara, Alessia Ventrella, Gabriella Siani, Tatiana Da Ros, Giovanna Iezzi, Amelia Cataldi, Antonella Fontana, Roberta Di Carlo, Susi Zara, Alessia Ventrella, Gabriella Siani, Tatiana Da Ros, Giovanna Iezzi, Amelia Cataldi, Antonella Fontana

Abstract

(1) Background: The aim of this study was to optimize, through a cheap and facile protocol, the covalent functionalization of graphene oxide (GO)-decorated cortical membrane (Lamina®) in order to promote the adhesion, the growth and the osteogenic differentiation of DPSCs (Dental Pulp Stem Cells); (2) Methods: GO-coated Laminas were fully characterized by Scannsion Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) analyses. In vitro analyses of viability, membrane integrity and calcium phosphate deposition were performed; (3) Results: The GO-decorated Laminas demonstrated an increase in the roughness of Laminas, a reduction in toxicity and did not affect membrane integrity of DPSCs; and (4) Conclusions: The GO covalent functionalization of Laminas was effective and relatively easy to obtain. The homogeneous GO coating obtained favored the proliferation rate of DPSCs and the deposition of calcium phosphate.

Keywords: calcium phosphate deposition; cortical membranes; covalent functionalization; graphene oxide.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Photographs of (A) pure Lamina, (B) Lamina functionalized with 3-aminopropyl triethoxysilane (APTES) (see Experimental Section 2.2), (C) Lamina enriched with 5 µg/mL graphene oxide (GO) and (D) Lamina enriched with 10 µg/mL GO.
Figure 2
Figure 2
Thermo-gravimetric analyses (TGA) of graphene oxide (black curve), bare Lamina (red curve), 5 µg/mL GO enriched Lamina (blue curve) and Lamina enriched with 10 µg/mL GO (green curve).
Figure 3
Figure 3
(A,D,G) Topographical, (B,E,H) Peak force error and (C,F,I) Three-dimensional Atomic Force Miscroscopy (AFM) images of bare Lamina (upper line), Lamina enriched with 5 µg/mL of GO (central line) and Lamina enriched with 10 µg/mL GO (bottom line).
Figure 4
Figure 4
Scansion electron microscopy (SEM) images of (A) bare Lamina, (B) APTES-treated Lamina, (C) 5 µg/mL GO-coated Lamina and (D) 10 µg/mL GO-coated Lamina. Magnification 3000×. Scale bar: 200 µm.
Figure 5
Figure 5
SEM images of Dental Pulp Stem Cells (DPSC)) cultured on bare Laminas (CTRL1), APTES-treated (CTRL2), 5 µg/mL GO-coated (GO5) and 10 µg/mL GO-coated (GO10) Laminas for 7 and 14 days. Magnification 3000×.
Figure 6
Figure 6
Alamar blue assay in DPSC cultured on bare Laminas (CTRL1), APTES-treated (CTRL2), 5 µg/mL GO-coated (GO5) and 10 µg/mL GO-coated (GO10) Laminas for 3, 7, 14, and 28 days. Forty Laminas were used for each experimental point, ten Laminas per experimental time. The histogram represents Alamar blue reduction percentage, data shown are the mean (±SD) of three separate experiments. Zero time % reduction Alamar Blue: 15.72%; * Day 7: GO5 and GO10 Laminas vs. control (CTRL1) Laminas p < 0.001; Day 14 control Laminas, GO5-coated and GO-10 coated Laminas vs. APTES-treated Laminas (CTRL2) p < 0.001.
Figure 7
Figure 7
Lactate Dehydrogenase (LDH) assay of DPSC cultured on bare Laminas (CTRL1), APTES-treated (CTRL2), 5 µg/mL GO-coated (GO5) and 10 µg/mL GO-coated (GO10) Laminas for 3, 7, 14, and 28 days. Forty Laminas were used for each experimental point, ten Laminas per experimental time. Released LDH is reported as percentage. Data shown are the mean (±SD) of three separate experiments. Zero time LDH release (%): 73.06 * Day 3: 10 µg/mL GO-coated Laminas (GO10) vs. control (CTRL1) p < 0.05; * Day 7: APTES-treated, 5 µg/mL GO-coated (GO5) and 10 µg/mL GO-coated Laminas (GO10) vs. control (CTRL1) p < 0.001; control Laminas, APTES-treated Laminas vs. 5 µg/mL GO-coated Laminas (GO5) p < 0.001; control Laminas, APTES-treated Laminas vs. 10 µg/mL GO-coated Laminas (GO10) p < 0.001; * Day 14, day 28: APTES-treated, GO10 Laminas vs. control (CTRL1) Laminas p < 0.005.
Figure 8
Figure 8
The histogram represents optical density (OD) values of solubilized calcium deposits (orange-red stained) obtained after Alizarin Red staining on bare Laminas (CTRL1), APTES-treated (CTRL2), 5 µg/mL GO-coated (GO5) and 10 µg/mL GO-coated (GO10) Laminas. Twenty Laminas were used for each experimental point, ten Laminas per experimental time. Data shown are the mean (±SD) of three separate experiments. * Day 21: APTES-treated Laminas vs. control (CTRL1) Laminas p < 0.005; * Day 28: control Laminas, APTES-treated, 10 µg/mL GO-coated Laminas (GO10) vs. 5 µg/mL GO Laminas (GO5) p < 0.005.

References

    1. Osteobiol by Tecnoss. [(accessed on 15 December 2018)]; Available online: .
    1. Rossi R., Rancitelli D., Poli P.P., Rasia Dal Polo R., Nannmark U., Maiorana C. The use of collagenated porcine cortical lamina in the reconstruction of alveolar ridge defects. A clinical and histological study. Minerva Stomatol. 2016;65:257–268.
    1. Jianguo L., Yanqun L., Haiyan L., Yufen X., Yongxiang Z., Jingxian L., Jianping W. Preparation, bioactivity and mechanism of nano-hydroxyapatite/sodium alginate/chitosan bone repair material. J. Appl. Biomater. Funct. Mater. 2018;16:28–35.
    1. Enayati-Jazi M., Solati-Hashjin M., Nemati A., Bakhshi F. Synthesis and characterization of hydroxyapatite/titania nanocomposites using in situ precipitation technique. Superlattices Microstruct. 2012;51:877–885. doi: 10.1016/j.spmi.2012.02.013.
    1. Kealley C., Elcombe M., van Riessen A., Ben-Nissan B. Development of carbon nanotube reinforced hydroxyapatite bioceramics. Physic B. 2006;385–386:496–498. doi: 10.1016/j.physb.2006.05.254.
    1. Novoselov K.S., Fal’ko V.I., Colombo L., Gellert P.R., Schwab M.G., Kim K. A roadmap for graphene. Nature. 2012;490:192–200. doi: 10.1038/nature11458.
    1. Kenry L.W., Loh K.P., Lim C.T. When stem cells meet graphene: Opportunities and challenges in regenerative medicine. Biomaterials. 2018;155:236–250. doi: 10.1016/j.biomaterials.2017.10.004.
    1. Balandin A.A. Thermal properties of graphene and nanostructured carbon materials. Nat. Mater. 2011;10:569–581. doi: 10.1038/nmat3064.
    1. Wang X.L. Proposal for a new class of materials: Spin gapless semiconductors. Phys. Rev. Lett. 2008;100:156404. doi: 10.1103/PhysRevLett.100.156404.
    1. Guazzo R., Gardin C., Bellin G., Sbricoli L., Ferroni L., Ludovichetti F.S., Piattelli A., Antoniac I., Bressan E., Zavan B. Graphene-Based Nanomaterials for Tissue Engineering in the Dental Field. Nanomaterials. 2018;8:349. doi: 10.3390/nano8050349.
    1. Bresson E., Ferroni L., Gardin C., Sbricoli L., Gobbato L., Ludovichetti F.S., Tocco I., Carraro A., Piattelli A., Zavan B. Graphene based scaffolds effects on stem cells commitment. J. Transl. Med. 2014;12:296. doi: 10.1186/s12967-014-0296-9.
    1. Ettorre V., De Marco P., Zara S., Perrotti V., Scarano A., Di Crescenzo A., Petrini M., Hadad C., Bosco D., Zavan B., et al. In vitro and in vivo characterization of graphene oxide coated porcine bone granules. Carbon. 2016;103:291–298. doi: 10.1016/j.carbon.2016.03.010.
    1. De Marco P., Zara S., De Colli M., Radunovic M., Lazović V., Ettorre V., Di Crescenzo A., Piattelli A., Cataldi A., Fontana A. Graphene oxide improves the biocompatibility of collagen membranes in an in vitro model of human primary gingival fibroblasts. Biomed. Mater. 2017;12:055005. doi: 10.1088/1748-605X/aa7907.
    1. Radunovic M., De Colli M., De Marco P., Di Nisio C., Fontana A., Piattelli A., Cataldi A., Zara S. Graphene oxide enrichment of collagen membranes improves DPSCs differentiation and controls inflammation occurrence. J. Biomed. Mater. Res. Part A. 2017;105:2312–2320. doi: 10.1002/jbm.a.36085.
    1. De Colli M., Radunovic M., Zizzari V.L., di Giacomo V., Di Nisio C., Piattelli A., Calvo Guirado J.L., Zavan B., Cataldi A., Zara S. Osteoblastic differentiating potential of dental pulp stem cells in vitro cultured on a chemically modified microrough titanium surface. Dent. Mater. J. 2018;37:197–205. doi: 10.4012/dmj.2016-418.
    1. Guo T., Cao G., Li Y., Zhang Z., Nör J.E., Clarkson B.H., Liu J. Signals in Stem Cell Differentiation on Fluorapatite-Modified Scaffolds. J. Dent. Res. 2018;97:1331–1338. doi: 10.1177/0022034518788037.
    1. Xie H., Chua M., Islam I., Bentini R., Cao T., Viana-Gomes J.C., Castro Neto A.H., Rosa V. CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells. Dent. Mater. 2017;33:e13–e21. doi: 10.1016/j.dental.2016.09.030.
    1. Engler A.J., Sen S., Sweeney H.L., Discher D.E. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–689. doi: 10.1016/j.cell.2006.06.044.
    1. Lee W.C., Lim C.H.Y.X., Shi H., Tang L.A.L., Wang Y., Lim C.T., Loh K.P. Origin of Enhanced Stem Cell Growth and Differentiation on Graphene and Graphene Oxide. ACS Nano. 2011;5:7334–7341. doi: 10.1021/nn202190c.
    1. Wei X.-Q., Hao L.-Y., Shao X.-R., Zhang Q., Jia X.-Q., Zhang Z.-R., Lin Y.-F., Peng Q. Insight into the Interaction of Graphene Oxide with Serum Proteins and the Impact of the Degree of Reduction and Concentration. ACS Appl. Mater. Interfaces. 2015;7:13367–13374. doi: 10.1021/acsami.5b01874.
    1. Tang L.A., Lee W.C., Shi H., Wong E.Y., Sadovoy A., Gorelik S., Hobley J., Lim C.T., Loh K.P. Highly wrinkled cross-linked graphene oxide membranes for biological and charge-storage applications. Small. 2012;8:423–443. doi: 10.1002/smll.201101690.
    1. Alberto B. Graphene: Safe or Toxic? The Two Faces of the Medal. Angew. Chem.-Int. Ed. 2013;52:4986–4997.
    1. Malvindi M.A., De Matteis V., Galeone A., Brunetti V., Anyfantis G.C., Athanassiou A., Cingolani R., Pompa P.P. Toxicity Assessment of Silica Coated Iron Oxide Nanoparticles and Biocompatibility Improvement by Surface Engineering. PLoS ONE. 2014;9:e85835. doi: 10.1371/journal.pone.0085835.
    1. Palermo E.F., Lee D.-K., Ramamoorthy A., Kuroda K. Role of Cationic Group Structure in Membrane Binding and Disruption by Amphiphilic Copolymers. J. Phys. Chem. B. 2011;115:366–375. doi: 10.1021/jp1083357.
    1. Riccio M., Resca E., Maraldi T., Pisciotta A., Ferrari A., Bruzzesi G., De Pol A. Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures. Eur. J. Histochem. 2010;54:205–213. doi: 10.4081/ejh.2010.e46.

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe