The Gemstone Cyborg: How Diamond Films Are Creating New Platforms for Cell Regeneration and Biointerfacing

Abstract

Diamond is a promising material for the biomedical field, mainly due to its set of characteristics such as biocompatibility, strength, and electrical conductivity. Diamond can be synthesised in the laboratory by different methods, is available in the form of plates or films deposited on foreign substrates, and its morphology varies from microcrystalline diamond to ultrananocrystalline diamond. In this review, we summarise some of the most relevant studies regarding the adhesion of cells onto diamond surfaces, the consequent cell growth, and, in some very interesting cases, the differentiation of cells into neurons and oligodendrocytes. We discuss how different morphologies can affect cell adhesion and how surface termination can influence the surface hydrophilicity and consequent attachment of adherent proteins. At the end of the review, we present a brief perspective on how the results from cell adhesion and biocompatibility can make way for the use of diamond as biointerface.

Keywords: NCD; NSCs; UNCD; adhesion; biointerfacing; cells; differentiation; neurons.

Figure 1

Schematic representation of the various steps of chemical vapour deposition (CVD) diamond growth on non-diamond substrates: (a) substrate surface enriched with diamond seeds, (b) seeded substrate exposed to diamond growth conditions, and (c) coalesced diamond film.

Figure 2

SEM micrographs showing the morphology of: (a) microcrystalline diamond; (b) nanocrystalline diamond (NCD); (c) ultrananocrystalline diamond (UNCD). (b) is reprinted with permission of Elsevier from Diamond and Related Materials, Vol. 15, O.A. Williams, M. Daenen, J. D’Haen, K. Haenen, J. Maes, V. Moshchalkov, M. Nesládek, D.J. Gruen, Comparison of the growth and properties of ultrananocrystalline diamond and nanocrystalline diamond, pp. 654–658 [23], ©Elsevier, 2006. (c) is reprinted with permission from Sumant, A.; Grierson, D.; Gerbi, J.; Carlisle, J.; Auciello, O.; Carpick, R., Physical Review B, 76, 235429, 2007, © American Physical Society, 2007 [24].

Scheme 1

Stages of in vitro mechanical cell adhesion, studied using epithelial cells and showing the main physical and biomolecular processes involved: electrostatic interaction in the sedimentation stage, integrin-mediation in the attachment/spreading stage, and production of heparan sulphate proteoglycane in the stage of stabilisation of the adhesion. * PDL = poly-D-lysine, an adhesion-promoting molecule, was added to the substrate; HSPG = heparan sulphate proteoglycane. Adapted with permission from Hong et al. [28].

Figure 3

NCD film with 200 μm wide H/O-terminated patterns: (a) optical (bright field) image prior to cell plating showing optically transparent and featureless surface, (b) SEM image prior to cell plating where bright stripes correspond to H-termination and dark stripes to O-termination of the diamond surface due to their opposite electron affinity. Fluorescent microscopy images of osteoblastic cells (SAOS-2) cultivated on the substrate, showing (c) the alignment cross for correlation of the surface termination micro-patterns with the cells, and (d) cell growth, after 2 days of incubation, on 200 μm H/O-terminated stripes on diamond films in McCoy’s medium supplemented with different foetal bovine serum (FBS) concentrations (0, 5, 10, and 15%). Reproduced from Rezek et al. [15] under a creative commons licence.

Figure 3

NCD film with 200 μm wide H/O-terminated patterns: (a) optical (bright field) image prior to cell plating showing optically transparent and featureless surface, (b) SEM image prior to cell plating where bright stripes correspond to H-termination and dark stripes to O-termination of the diamond surface due to their opposite electron affinity. Fluorescent microscopy images of osteoblastic cells (SAOS-2) cultivated on the substrate, showing (c) the alignment cross for correlation of the surface termination micro-patterns with the cells, and (d) cell growth, after 2 days of incubation, on 200 μm H/O-terminated stripes on diamond films in McCoy’s medium supplemented with different foetal bovine serum (FBS) concentrations (0, 5, 10, and 15%). Reproduced from Rezek et al. [15] under a creative commons licence.

Figure 4

Comparison of: (a) adhesion of neural stem cells (NSCs) (from a seeding density of 3.5 × 10⁴ cells/cm²) to a polystyrene (PS) Petri dish (black bars) and to H-UNCD films (grey bars), with data expressed as mean ± standard deviation (n = 4); (b) length of filopodia (100 nm in width) of NSCs cultured on polystyrene (black bars) and H-UNCD films (grey bars), in supplemented medium, at 8, 12, and 36 h of culture. More than 30 cells were randomly picked and measured for each sample by an unbiased investigator who was not informed of the nature of each sample. SEM micrographs, at low (inset) and high magnification of NSCs cultured for 8, 12, and 36 h, on: (c–e) polystyrene, showing short filopodia with few protrusions that do not spread far from the main cell body; (f–h) H-UNCD, showing numerous, long and complex filopodia that extend away from the main cell body. Significance levels: *** p < 0.001 [6].

Scheme 2

Schematic representation of the role of H-UNCD films in mediating differentiation from NSCs. Adsorbed fibronectin on H-UNCD surface activates integrin, Fak, and Erk1/2 pathways which trigger NSC differentiation into neurons [6].

Figure 5

Adhesion and growth of 17-day rat embryo hippocampal neurons and glial cells on (A) NCD substrates coated with an adhesion-promoting layer of poly-D-lysine and laminin, and (B) virgin NCD substrates, as shown by confocal microscopy images with immunostaining. Cells were stained with anti-neurofilament antibodies (NF, giving neurons a fluorescent green colour), 4′,6-diamidino-2-phenylindole (DAPI, marking the DNA-rich nucleus of neurons with blue) and antibodies for glial fibrillary acidic protein (anti-GFAP, making glial cells appear in red). In each image, scale bar = 20 μm. Adapted from Ojovan et al. [41] under a creative commons licence.

Figure 6

Neural growth in function of diamond surface morphology: (a) average distance between sharp peaks on two adjacent crystallites; values represent the average of 20 distances on 4 microscopic fields from 2 samples. (b) Number of cell nuclei present on each type of substrate, compared in function of the number of cells growing on the control substrate (polished non-doped diamond) that has been normalised to 1. (c) Percentage of area (mean ± s.d., n = 3) devoid of neurites for each type of substrate. * p < 0.05, *** p < 0.001. Adapted from Nistor et al. [40] under a Creative Commons licence.

Figure 7

SEM image of the 3D-nanostructured BDD MEA, (A) viewed from the side to show the interface of the BDD and the 3D-nanostructured BDD, and (B) viewed from the top to show the topography (Scale bar = 600 nm). Adapted from Piret et al. [46] under a Creative Commons licence.