Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films

Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH4:H2 gas mixture, the B∶C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from >10 MΩ (undoped films) to 55 kΩ, 0.6 kΩ, and 0.3 kΩ (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000±14,000 and 152,000±10,000 cells/cm2, respectively, compared to 113,000±10,000 cells/cm2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells

Surface morphology of NCD samples without boron doping (A), with boron 133 ppm (B), 1000 ppm (C) and 6700 ppm (D).

<p>Field emission scanning electron microscope (e_Line, Raith). Scale bar is 200 nm.</p

Neutron depth profiling of boron over doped NCD samples.

<p>Neutron depth profiling of boron over doped NCD samples.</p

Number of MG 63 cells on day 1, 3 and 7 (A, C, E), their spreading area (D) and their growth dynamics (B) on a standard polystyrene cell culture dish (PS), undoped NCD films (B_0) and NCD films doped with 133, 1000 and 6700 ppm of boron (B_133, B_1000 and B_6700, respectively).

<p>Mean ± S.E.M. from 3 experiments; each included 32 microphotographs (day 1 and 3) and 18 measurements in a hemocytometer (day 7) per experimental group). ANOVA, Student-Newman-Keuls method. Statistical significance: I, II, III, IV, V: <i>p</i>≤0.05 compared to the group labelled with the same Roman number.</p

The cell population doubling time of MG 63 cells between days 1 and 3 (DT_1–3), days 3 and 7 (DT_3–7) and days 1 and 7 (DT_1–7) after seeding on polystyrene culture dishes (PS) and NCD films doped with 0, 133, 1000 or 6700 ppm of boron.

<p>Mean ± S.E.M. from 3 experiments (in total, 9 measurements for each experimental group and time interval). ANOVA, Student-Newman-Keuls Method. Statistical significance: <b>^{I, II, V}</b>: <i>p</i>≤0.05 compared to polystyrene, undoped NCD and NCD doped with 6700 ppm of B, respectively.</p

Immunofluorescence staining of talin in MG 63 cells on day 3 after seeding on microscopic glass coverslips (A), undoped NCD (B), NCD films doped with boron in concentrations of 133 ppm (C) 1000 ppm (D) and 6700 ppm (E).

<p>The cell nuclei are counterstained with propidium iodide. Olympus IX 51 epifluorescence microscope, DP 70 digital camera, obj. 100×, bar = 10 µm.</p

Raman spectra of undoped and boron-doped NCD samples.

<p>Raman spectra of undoped and boron-doped NCD samples.</p

Microscopic images of surface potential variations as detected by Kelvin force microscopy (left column), and surface topography as detected by atomic force microscopy (right column) on NCD samples without boron doping (A) and with nominal boron doping of 133 ppm (B), 1000 ppm (C), and 6700 ppm (D).

<p>The AFM oscillation amplitude was 100 nm, the setpoint was 50%. The KFM lift height was 30 nm. The detection frequency was 75 kHz.</p