Viability and proliferation of A549 cell line on the surface of micro-, nano- and ultrananocrystalline diamond films grown by HFCVD with tailored gases

Abstract This article describes key material science/technology issues to implement polycrystalline diamond scaffolds to enable processes for biological cells growth relevant for using cells grown in the laboratory for the treatment of human biological conditions. Issues investigated include 1. Synthesis/characterization of microcrystalline diamond (MCD), nanocrystalline diamond (NCD) and transformational ultrananocrystalline diamond (UNCD) coating-based scaffolds. Diamond films were grown on silicon substrates using the hot filament chemical vapor deposition (HFCVD) technique, by which filaments heated to ∼2,300 °C induce cracking of CH4 molecules into C atoms/CHx (x = 1, 2, 3) radicals, growing diamond films via chemical reaction on the substrates’ surface. MCD and NCD + films were grown flowing the H2/CH4 gas mixture. NCD- and UNCD films were grown using the Ar/CH4 gas mixture plus H2 flux (73.5%, 49%, and 9.8%), which for high fluxes, induced increased the concentration of H-containing trans-polyacetylene (T-PA) molecules in UNCD films’ grain boundaries, impacting biological performance. 2. Studies of viability and proliferation of human lung carcinoma cell line (A549) grown on surfaces of MCD, NCD, and UNCD films, using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which showed no significant difference in cell proliferation among the MCD, NCD, and UNCD films.


Introduction
Extensive R&D is being done to investigate potential biocompatible materials to enable new generations of implantable medical devices/prostheses/artificial cells into the human body. Materials investigated and/or currently used in implantable medical devices, including metals (eg, gold, iridium, platinum, and titanium), polymers (eg, polytetrafluoroethylene\ and polyurethane), and ceramics (eg, zirconia and zirconia-titanium alloy) [1].
Ti-alloys (Ti-6Al-4V) are being extensively used for dental implants (DI) [1,2], which exhibit ∼15% failure worldwide in the first 4-5 years due to oral fluids that induced chemical corrosion [2,3]. Other prostheses (eg, hips and knees) present similar problems including chemical corrosion [4]. Metals, in general, develop oxide layers upon atmosphere exposure, resulting in body fluid-induced oxide particles ejection, inducing natural human cells death, inflammation of tissues, and other biological degradation effects, as for Ti-alloys DIs [3,5].
Key application of polycrystalline diamond films, relevant to this article, relies on their biocompatibility and surface chemistry, enabling growth of cells and negligible toxicity [see, eg, refs. [8,21] and references there in). These properties are based on the unique sp 3 C atom bonds' structure and chemical surface termination. A relevant process for human cells' growth on diamond surfaces is the functionalization to produce O or H atom termination. For specific biomedical implantable devices, requiring human cells adhesion is critical to produce hydrophilic surfaces via O-plasma processing, inducing O atoms surface termination [17]. Studies revealed that adsorbed biological materials, such as buffer layers, can enhance cells' adhesion on the diamond surface [22]. Alternatively, H-terminated diamond surface can be used for sensors or electronic devices [18][19][20] but exhibit high water drop contact angle, resulting in hydrophobicity and negligible adherence of cells, like blood cells interacting with artificial heart valves or stents, which may result in life threatening thrombus formation [8] or coli bacterium interaction with implantable medical devices [21].
Based on the information presented above, this article is focused on describing results related to biological viability and proliferation of A549 cell line on MCD, NCD, and the UNCD films surfaces, for films grown with different H 2 gas flow concentration, tailoring chemical treatments of diamond films' surfaces.

Diamond films growth processes
The diamonds films used for cell growth were grown on several silicon (Si) substrates in a Blue-Wave HFCVD system. The Si substrates were seeded with nanocrystalline diamond particles on the surface to induce the diamond film growth using the well know seeding process extensively described in the literature (see reviews in references [6,9] and patent [7]). The parameters for the diamond films' growth process in the HFCVD system were tailored for growing MCD, NCD, and UNCD films based on previous research [12] (see Table 1).

Assay of mitochondrial viability (MTT assay)
Cell viability was determined using the MTT assay (3 -(4,5-dimethylthiazol-2-yl) 5 -(3-carboxymethoxyphenyl) −2 -(4-sulfophenyl) −2H-tetrazolium), which corresponds to a colorimetry test based on the reduction in MTT to formazan crystals by mitochondrial dehydrogenase enzymes. Living cells reduced MTT to formazan, which is quantified by measuring absorbance. The A549 cell line was cultured in a culture flask, and when the growth reached 100% of confluence, the cells were detached from the culture flask by trypsinization, and the cell suspension was counted in Neubauer chamber, and it was adjusted to a concentration 100,000 cells/ ml. Then, this cell suspension was deposited, fully covering the surfaces of MCD, NCD, and UNCD film's grown on Si substrates with an area of 2.25 cm 2 . Previously, the cells were sterilized by autoclave at 120 °C for 20 min and placed in 12-well plates. The cells were cultured in each well in a volume of 1.5 ml of DMEM, supplemented with 10% FBS, and incubated at 37 °C with 5% CO 2 during 48 h. Cells with the same density were seeded in empty wells without any materials, as control. The cells were removed using trypsin, followed by centrifugation at 1,500 rpm (348 x g), and 4 °C for 10 min. Some wells with cells were prepared for counting, using trypan blue. Other cells were used to perform the MTT assay, involving 5 µl of MTT (0.5 mg/ml in a Table 1. Parameters for the growth of mcD, ncD, and uncD films. the ncD+, ncD-mid, and ncD-notation refers to the ncD's grain sizes, which are controlled by the flow of H 2 gas in the gas mixture. medium) added to the well. Cells grown on the MCD, NCD, and UNCD surfaces and the control surface were incubated for 6 h at 37 °C. Finally, formazan crystals were solubilized in acidified isopropanol and the absorbance of each sample was measured at 570 and 655 nm. The data from this study are reported as the mean ± standard deviation (SD) of three independent assays. The significance of the data between experimental groups was determined by using the ANOVA method, followed by Tuckey-Kramer-type analysis, using a NCSS software 2007 version. Values of p ≤ 0.05 were considered statistically significant. The results were plotted using a GraphPad prism software version 8.0.2. A detailed schematic of the cell culture process is shown in Figure 1.
The cell viability was also measured by counts in a Neubauer chamber. The protocol used for counting A549 cells include (1) A549 cells growth on diamond and silicon surfaces; (2) after 48 h, cells were removed by trypsin and centrifuged as described above; (3) cells were counted in a Neubauer chamber, involving optical microscopy; (4) A549 cells were counted over each of the four grid blocks with trypan blue 1:1 diluted. The results are shown in Table 2 in Section 3.2.
The surface topography of the diamond films was characterized in a Zeiss Supra 40 scanning electron microscope (SEM) operated at 15.00 kV for MCD and NCD films and 10.00 kV for UNCD films. The chemical bonding of diamond films was studied in a LabRAM HR Raman spectrometer equipped with an Ar ion laser (λ = 532 nm) as an excitation source. Optical microscopy images of the adhered cells on the diamond films and silicon were obtained using the microscope of the Raman spectrometer. Prior to observation, the 48 h incubated cells grown on the surface of the MCD, NCD, and UNCD films were washed and incubated during 10 min in physiological solution and fixed with 4% formaldehyde solution. Finally, the MCD, NCD, and UNCD films' surfaces were observed using the optical microscopy. Figure 2 shows typical normalized Raman spectra of MCD, NCD, and UNCD films. Raman spectra of MCD and NCD + films are shown in Figure 2a, which reveal the fingerprint of diamond with the signal of C-C sp 3 chemical bonds at 1332 cm −1 and the prominent G-band at ∼1567 cm −1 associated to C-C sp 2 chemical bond of C atoms in the gran boundaries (see review in ref. [5]). The difference between the high of the diamond 1332 cm −1 peak of MCD and NCD films is correlated with the grain size [23]; the only difference between both films were the growth time (8 h for MCD and 4 h for NCD + films). The Raman spectra shown in Figure 2b, of the three films grown with different gas flows, correspond to the NCD-mid film grown with 73.9% H 2 , NCD-film grown with 48% H 2 and UNCD film grown with 9.8% H 2 . The three films show two main bands, the 1344 cm −1 signal that is associated to the D-band, encapsulating the peak at 1332 cm −1 and the Raman signal at ∼1580 cm −1 , corresponding to the G-band which has been shown, for microcrystalline diamond films, to correlate with sp 2 chemical bonds of C-C atoms in the grain boundaries of the films, but not in a graphite structure [5,23]. In addition, two Raman signals at ∼1164 cm −1 and ∼1489 cm −1 , respectively, have been identified as correlated with trans-polyacetylene (T-PA)/C-H molecules, with chemical bonds associated with C-C stretching and C = C stretching in T-PA molecules, respectively [17,18]. The intensities of the Raman signal related to diamond at 1332 cm −1 are reduced in correlation with the grain size reduction from MCD to UNCD films [18]. The Raman spectra of UNCD films grown with 9.8% of H 2 show the maximum G-band near ∼1580 cm −1 with more C-C sp 2 chemical bonds due to larger grain boundaries network. In addition, as the gas flow increase, during film growth, the Raman peak related to the G-band moves to ∼1548 cm −1 (see Figure 2b, middle for NCD-) with more C-C sp 2 chemical bonds and increase in the Raman signal of T-PA molecules correlated with ∼1164 of C and ∼1489 cm −1 . For NCD-mid film with major H 2 gas flow concentration, during the film growth, the diamond peak at ∼1332 cm −1 can still be seen, but with very low intensity (Figure 2b, top). The NCD-mid with 73.9% of H 2 and NCD-with 48% of H 2 films show increased Raman signal correlated with T-PA molecules and C-C sp 2 chemical bonds in grain boundaries [24][25][26]. Figure 3 shows SEM images of the surface morphology of MCD, NCD+, NCD-mid (grown with 73.5% H 2 (75 sccm)), NCD-(grown with 49% H 2 (50 sccm)), and UNCD (grown with 9.8% H 2 (25 sccm)) films (see Table 1 for detailed gas flow conditions, including flows for CH 4 and Ar gases). The surfaces of MCD and NCD + films exhibit high and medium surface roughness (Figures  3(a) and 3(b), respectively). The surfaces of the NCD-mid and NCD-films exhibit smoother surfaces (Figures 3(c) and 3(d), respectively). The surface of the UNCD films is the smoothest, due to the extremely small grain sizes (3-10 nm) (Figure 3(e)). The thickness of the different diamond films was measured by cross-section SEM (see inserts in Figures 3(a-e), and thickness values listed in Table 1 and plotted in Figure 3(f).  Figure 3 for grain size information). all spectra were obtained by shining a laser beam with wavelength λ = 535 nm on the surface of the films; the peak at 1332 cm −1 is the fingerprint for diamond, which can be seen with visible laser wavelength λ = 532 nm only for ncD+, ncD-mid, and mcD films; ncD-and uncD films do not reveal the 1332 cm −1 peak with laser beam of λ = 532 nm because of the extreme small grains (uV laser (λ = 244 nm) is necessary to reveal the diamond peak).

Measurement of cell proliferation via MTT analysis and cell adherence observation via optical microscopy on diamond surfaces and control Si surface
Investigation of cell viability and proliferation on MCD, NCD, and UNCD films were determined using the MTT assay. Optical microscopy provided valuable information to confirm the growth of A549 cells surfaces and morphology on the diamond surfaces after 48 h (Figure 4). The optical images of cell growth on the MCD, NCD+, NCD-mid, NCD-, and UNCD films showed good cell attachment and proliferation, with morphological normal elongated shape cells and formation of cells' cumulus. The roughness of diamond films observed by SEM in Figure 3 apparently does not induce differences correlated with affinity between A549 cells and diamond films. The Si surface shows only small remains of cells with no proliferation (Figure 4(f)).
The total numbers of cells were counted after 48 h. The results of MTT assay are shown in Figure 5. Cell culture on NCD-film, grown with 49% of H 2 , exhibits the highest percent of cell proliferation on 2.25 cm 2 . Figure 5 shows that MCD, NCD, and UNCD films exhibit similar high cell growth and proliferation ∼10x higher than for Si surfaces. The quantitative data shown in Figure 5 indicate that diamond films provide excellent scaffolds for growing human cells and eventually make them differentiate into other human cells, via electrical excitation, as the Auciello group is now demonstrating in new experiments, to be reported in a future article. The hypothesis to explain the excellent biological performance of diamond surfaces is that the diamond films used for scaffolds are made of C atoms with unique chemical bonds and tailored surface chemical termination, enabling biological cells to recognize C atoms on the surface of the diamond films as C atoms in cells. The results shown in Figure 5 can be compared positively with the results from other groups on interactions of cells with nanodiamond particles with dimensions (3-5 nm) [27,28] identical to the grain size of UNCD films [6][7][8]. Table 2 shows the values of cell concentration after 48 h measurements in a Neubauer chamber with the

Conclusions
Key conclusions, based on the fundamental and applied materials and biological related science described in this article, are 1. Microcrystalline diamond (MCD-grains sizes ≥ 1 µm), nanocrystalline diamond (NCD-grains sizes 20-100s nm), and ultrananocrystalline diamond (UNCD-grain sizes 3-8 nm) films can be grown with very good chemical and biocompatible properties, using the HFCVD technique, to provide scaffolds for investigating the growth and proliferation of A549 cells, demonstrating the feasibility of using the polycrystalline diamond surfaces for studies of other biological cells' growth. 2. The H 2 flow during the growth of UNCD to NCD films appears to affect the presence of trans-polyacetylene (T-PA) molecules in the grain boundaries, as revealed by the Raman signal correlated with T-PA molecules. However, the T-PA molecules' concentration appear not to make a substantial difference in the proliferation of cells on the surface of all MCD, NCD, and UNCD films, as shown in Figure 5. 3. The MTT assay, using A549 cells, showed very small difference in the cell growth and proliferation on the surfaces of the MCD, NCD, and UNCD films, and all showed about 10x higher cell proliferation than Si surfaces, which may be explained by the fact that the diamond surface provides C atoms with sp 3 -diamond bonds and chemical surface termination enabling superior cells growth and proliferation. 4. The negative results about the cell growth on the Si surface support the results obtained in the measurements by the MTT assay and counts in the Neubauer chamber, because the A549 cells' counts, corresponding to the top surface of diamond films, are not affected by the Si substrate underneath the diamond films. 5. The surface roughness and topography exhibit some correlation with the cell proliferation and rate growth. MCD and NCD films show major rate growth cell.
Work in progress will provide more valuable information towards exploring growing embryonic stem cells and induced differentiation towards other human cells, via electrical excitation.