Nanocrystalline diamond (NCD) coatings combine a very low surface roughness with the outstanding diamond properties, such as superlative hardness, low self-friction coefficient, high wear and corrosion resistance, and biotolerance, which are ideal features for applications in medicine (knee and hip replacement) and surgical tools. The present work presents a comprehensive study of the cytotoxicity and biocompatibility of NCD films grown by hot-filament chemical vapour deposition (HFCVD) technique, aiming such future applications. Cytotoxicity was evaluated in vitro by seeding human gingival fibroblasts on the NCD surface for 14 days, while specific biocompatibility was assessed on samples seeded with human bone marrow-derived osteoblasts during 21 days. The NCD coatings proved to be noncytotoxic in the preliminary human gingival fibroblast cell cultures, as denoted by a notable sequence of cell attachment, spreading, and proliferation events. In the specific biocompatibility assay envisaging bone tissue applications, NCD coatings induced human osteoblast proliferation and the stimulation of differentiation markers, compared to standard polystyrene tissue culture plates.
Diamond has been one of the most desired
and investigated materials in the past years. From an extensive list of superlative
properties, the high hardness, the chemical inertness, the high thermal
conductivity, and the high optical transparency are just a few examples of its
remarkable nature. Applications such as cutting tools, abrasives, structural
components, heat sinks, bearings, and optical windows (X-ray, IR, and laser
windows) are the proof that diamond has a wide-ranging impact in several fields.
Diamond coatings with nanosized crystallites, the so-called nanocrystalline
diamond (NCD), present a great potential to become a serious challenging
technology in new application areas like biomedicine and biotechnology. NCD
combines surface smoothness with high corrosion resistance and biotolerance,
which are ideal features for applications in medicine onto surgical tools and medical
implants. For example, joint implants coated with NCD can take benefit of its
protective character. The NCD coating acts as a selective protective barrier
between the implant and the human environment, preventing the release of
metallic ions into the body [
Nowadays, a new generation of biomaterials,
able to control the biological response with precision, is arisen. NCD is also
included in this recent group of materials and can be used as a template for
the immobilization of active molecules for biological applications or for
biosensoric applications [
Despite all the referenced works report
the study of NCD bioproperties, the biocompatibility of this coating is not yet
full assessed. NCD films can differ to some extent in their surface and bulk
properties depending on the deposition method and parameters applied [
The present work reports the in vitro study of the citotoxicity and biocompatibility of NCD films grown on
Si3N4 substrates by hot-filament chemical vapour deposition (HFCVD) technique, using
Ar-CH4-H2 gas mixtures, considering their future
application as a coating for joint implants (knee and hip replacement). Si3N4 ceramics are used as substrates because they are not cytotoxic [
In this work, cytotoxicity was evaluated by seeding NCD coatings with human gingival fibroblasts while specific biocompatibility was assessed by the characterization of the seeded samples with human bone marrow-derived osteoblasts.
NCD
coatings were produced by the HFCVD technique. Dense Si3N4 ceramics substrates were prepared
according to a processing route that can be found elsewhere [
The NCD
deposition conditions were as follows:
NCD as grown: SEM cross-sectional (a) and AFM scan (b) views.
Primary
cultures were obtained by culturing explants of gingiva from a patient
undergoing a third molar extraction for orthodontic reasons. Informed consent
to use this biological tissue that would be otherwise discarded was obtained.
The tissue was washed in phosphate buffer saline (PBS), cut into small pieces,
and cultured in
Human bone marrow, obtained from orthopaedic surgical
procedures after patient-informed consent, was cultured in the same
experimental conditions as those used in the fibroblast cell cultures. Primary
cultures were maintained until near confluence (10–15 days) and, at this stage,
adherent cells were enzymatically released (trypsin–EDTA solution) and seeded at a density of
Control cultures and seeded material samples were characterized at days 3, 7, 14, and 21 for cell viability/proliferation (MTT assay), total protein content, alkaline phosphatase (ALP) activity, and observation by SEM and CLSM to evaluate cell morphology and matrix mineralization.
MTT assay (reduction of 3-[4,5-dimethylthiazol-2-yl]–2,5-diphenyltetrasodium bromide to a purple formazan reaction product by living cells) was used to estimate cell viability/proliferation. Cultures were incubated with
Culture samples were washed twice in PBS, frozen at −20°C,
and evaluated at the end of the culture time. The total amount of protein
present in the cultures was assayed by the Lowry’s method with bovine serum
albumin used as a standard. The results were expressed as
For SEM/EDS observation (JEOL JSM 6301F, equipped with an X-ray energy dispersive spectroscopy voyager XRMA system, Noran Instruments), cultures were fixed with 1.5% glutaraldehyde in 0.14 M sodium cacodylate buffer (10 minutes, pH 7.3), dehydrated in graded alcohols, critical-point dried, and sputter-coated with gold.
For CLSM assessment, fibroblast and osteoblast cell cultures were fixed in 3.7% paraformaldehyde (15 minutes)
followed by incubation in albumin (
Three experiments
were performed for each period of culture evaluation. The results are shown as
the arithmetic mean ± the standard deviation (±SD). Analysis of the results was
carried out using the nonparametric Kruskall-Wallis test, with a significance
level of
The biological profile of Si3N4 ceramics, used as
substrate material for the NCD coating, was addressed in previous in vitro and
in vivo studies. Human osteoblast-like cells cultured on polished Si3N4 ceramics showed enhanced proliferation and osteocalcin production compared to
standard polystyrene culture plates [
Results
regarding the behaviour of human fibroblast (HG) cells cultured on NCD coatings
are shown in Figures
Cell viability/proliferation of human gingival fibroblast cells cultured on NCD-coated Si3N4 substrates for 14 days, estimated by the MTT assay. ∗ statistically different from the control.
Time-course behaviour of human gingival fibroblast cell cultures grown over NCD-coated Si3N4 substrates for 14 days. CLSM (phalloidin and propidium iodide labelling): 6 hours (a), 12 hours (b), and 24 hours (c). SEM: 1 day (d) and 14 days (e), (f).
MTT assay
was used to assess cell viability/proliferation (Figure
Figures
Fibroblast
cell cultures established from normal tissues are a useful tool regarding the
evaluation of the cytotoxicity profile of medical devices, due to their
representative behaviour regarding the fibroblast cell population [
Results
showed that the NCD films allowed the adhesion and proliferation of human
fibroblast cells with the reproduction of the typical morphological features
and cell growth pattern expected for this cell type [
The
clinical success of a bone tissue implant relies on its osseointegration reflected
by a proper bone metabolism at the bone/implant interface. This represents a
dynamic process involving the migration of osteoprogenitor cells from the
adjacent bone marrow and the subsequent adhesion to the material surface, and
their proliferation and differentiation with the production of a mineralised
matrix around the implant material. In this work, NCD coatings were seeded with
first-passage human bone marrow (HBM) cells cultured in the presence of
ascorbic acid,
Cell viability/proliferation (a), total protein content (b), and ALP activity (c) of human bone marrow osteoblast cells grown on NCD-coated Si3N4 for 21 days. ∗ statistically different from the control.
Time-course behaviour of human bone marrow osteoblast cell cultures grown over NCD-coated Si3N4 substrates at 7 days ((a), (d)), 14 days (b), (e), and 21 days (c), (f). CLSM: (phalloidin and propidium iodide labelling, (a), (b); plus calcein staining, (c) and SEM (d)–(f) photographs. For comparison, the cell behaviour observed on standard polystyrene culture plates is also shown (SEM): (g), (h), (i), respectively, for 7, 14, and 21 days).
Matrix mineralization by human bone marrow osteoblast cell cultures grown over the NCD films at day 21. SEM: low magnification (a), detail of the mineralised structures (b), EDS spectrum of the mineralised deposits (c).
HBM
cells grown in control conditions proliferated during the first two weeks presenting
a high cell growth rate during the first week, as evident
in the MTT assay (Figure
CLSM and SEM (Figures
The present observations are in line with recent
studies on the in vitro osteoblastic
biocompatibility of NCD-coated surfaces that reported favourable results with
osteosarcoma SaOS-2 cells. The absence of cytotoxicity of a composite of NCD
and amorphous carbon was observed by SEM [
According to a variety of studies, osteoblasts recognise
differences in nanometer range compared with conventional topographies. Webster
et al. found increased functions of osteoblasts on nanophase compared to
conventional ceramics, polymers, carbon nanofibers, metals, and composites of
these materials [
Results obtained under this in vitro study strongly suggest that the prepared NCD coating presented improved features for osteoblast proliferation and function. In vivo experiments in animals, including histological studies and push-out strength measurements, are now imperative for a complete characterization of NCD as a biomaterial.
Hot-filament CVD grown nanocrystalline diamond (NCD) coatings provided a suitable surface for cell attachment, spreading, and proliferation, as assessed by the behaviour of human gingival fibroblast cells in a nonspecific cytotoxicity assessment. In addition, the seeded film allowed the reproduction of the typical morphological features and pattern of cell growth of this system, further suggesting a lack of cytotoxicity. In an osteoblastic biocompatibility assay, NCD coatings elicited an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared to standard polystyrene tissue culture plates. These results suggest the potential of NCD as a coating for joint implant applications. The unique mechanical properties and very low surface roughness of NCD support excellent biotribological behaviour at the prosthesis sliding contact. On the other side, by simulating the nanometric features of the bone tissue and by inducing bone growth, NCD coatings are able to play a proper osseointegration role.
M. Amaral acknowledges FCT for the Grant SFRH/BPD/ 26787/2006. The work was carried out with financial support from FCT Project POCI/CTM/45423/2002. Cell culture studies were performed at FMDUP and supported by this institution. The authors would like to thank Paula Sampaio (IBMC) for assistance with CLSM and Emanuel Carvalho Monteiro for gently preparing the samples for SEM observation.