# Nanocrystalline Diamond as a Coating for Joint Implants: Cytotoxicity and Biocompatibility Assessment

Authors: Margarida Amaral; Pedro S. Gomes; Maria A. Lopes; José D. Santos; Rui F. Silva; Maria H. Fernandes
Journal: Journal of Nanomaterials
 (2008)

---

## Abstract

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.


---

## Body

## 1. Introduction

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 [1]. NCD presents the highest resistance to
bacterial colonization when compared to medical steel and titanium [2]. This
property is very important since infection due to microbial colonization of the
implant surface may lead to implant rejection. In addition, the high wear resistance
and the low coefficient of friction of NCD allow the reduction of the amount of
wear debris generated during the joint functioning, increasing the life of the
prosthesis [3, 4]. Even more, in this case, the residues formed due to wear are
diamond particles, which are completely harmless initiating little or no
adverse reactions from human monocytes and polymorphonuclear leukocytes [5–7].
In fact, recently, it was shown that diamond particles appear to possess high
bioactivity at the molecular level, presenting antioxidant and anticarcinogenic
properties [8].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 [9–14]. One example is the functionalization of NCD
with bone morphogenetic protein-2 (BMP-2) creating a biomimetic coating that
results in improved osseointegration, which is a powerful strategy in tissue
engineering and in the context of bone tissue regeneration [15]. The NCD
surface can also be modified with the linking of an antibody, human
immunoglobulin G (IgG), which provides biomolecular recognition capability, turning
out a biologically sensitive field-effect transistor (Bio-Fet) [14].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 [9].
Besides, biocompatibility refers to the ability of a material to execute a
specific application with an appropriate host response [16]. Therefore, for
each type of NCD and respective application, the biocompatibility must be
assessed considering adequate in vitro and in vivo biological systems to
evaluate specific cell and tissue response.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 [17–19] and provide
an excellent film adhesion due to a small thermal expansion coefficient
mismatch with diamond. The HFCVD is a simple and low-cost method, able to coat
complex shapes and internal surfaces, like the femoral head and acetabular cup
of hip prosthesis. Biotribological tests for checking the mechanical
suitability of NCD as a coating for joint implants have already been made by the
present group [3, 4].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.


## 2. Materials and Experiments

### 2.1. NCD-Coated Samples

NCD
coatings were produced by the HFCVD technique. Dense Si3N4 ceramics substrates were prepared
according to a processing route that can be found elsewhere [20]. Prior to
deposition, the disc-shaped samples (diameter = 10 mm, thickness = 3 mm) were
ground and polished with 15 μm diamond slurry and submitted to a 1-hour ultrasonic bath in 1 μm diamond powder suspension in n-hexane (1 g/100 mL), followed by
ultrasonic cleaning with ethanol for 10 minutes.The NCD
deposition conditions were as follows:Ts = 750°C (substrate
temperature), Tf = 2300°C (tungsten filament temperature), Ar/H2  = 0.1, CH4/H2  =  0.04, P=50 mbar (total gas pressure), F=50 mL⋅min−1 (total gas flow), td=2hours (deposition time). The HFCVD deposition
method enabled an NCD coating of 2 μm in thickness, that is, a growth rate of about 1μm⋅hr−1.
Figure 1(a) gives a representative cross-section of the
film/substrate interface as taken by scanning
electron microscopy (SEM, Hitachi S4100). The typical surface microstructure of the NCD coatings is
depicted in the atomic force micrograph
(AFM, Digital Instrument Multimode IIIa) in Figure 1(b). The diamond
nanocrystals, with an average size of 28 nm [21], are agglomerated as
micrometric clusters, leading to a root-mean-square roughness (RMS) value of 68
± 5 nm (from AFM 100×100μm scans). A previous work demonstrated that the NCD coating supported a
normal load of 400N without spalling-off and presented an interfacial crack
resistance of 6.8 Nmm−1 [22]. Before being
seeded, the NCD-coated Si3N4 samples were washed with
ethanol in an ultrasonic cleaner and sterilized by autoclaving.NCD as grown: SEM cross-sectional (a) and AFM
scan (b) views.
(a)(b)


### 2.2. Cell Cultures

#### 2.2.1. Human Gingival Fibroblast Cells

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 inα-Minimal Essential Medium (α-MEM) containing 10% fetal bovine serum,  50μg⋅mL−1 ascorbic acid, 50μg⋅mL−1 gentamicin, and 2.5μg⋅mL−1 fungizone at 37°C, in a humidified atmosphere of 5% CO2 in air.  Cell outgrowth from the tissue
explants was observed 1 to 2 weeks after the beginning of the incubation. Cultures
showed a high proliferation rate and reached confluence in approximately 1 week,
with cells oriented along parallel lines, a typical feature of this culture
system [23]. Primary cultures were subcultured (0.05% trypsin, 0.25% EDTA) at
70–80% confluence.
First-passage human gingival (HG) cells were cultured (104cells⋅cm−2)
for 14 days in standard plastic culture plates and on the surface of the NCD
films. The medium was changed every 2-3 days. Cultures were characterized at 3,
7, and 14 days for cell viability/proliferation (MTT assay). In addition,
cultures were observed by confocal laser scanning microscopy (CLSM) and
scanning electron microscopy (SEM) to assess cell morphology during cell
adhesion to the substratum (first 24 hours) and throughout the culture time.


#### 2.2.2. Human Bone Marrow Cells

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 of104cells⋅cm−2 in standard polystyrene culture plates and on the
surface of the NCD films. Control and seeded material samples were cultured for
21 days in the experimental conditions described above, with the culture medium
further supplemented with 10 mM β-glycerophosphate and 10 nM dexamethasone.
All the experiments were performed in the first subculture since the sequential
passage of bone marrow osteoblastic cells fallout in a progressive loss of the
osteoblast phenotype [24].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.


### 2.3. Biochemical and Microscopy Assays

#### 2.3.1. Cell Viability/Proliferation

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 with0.5μg⋅mL−1 of MTT for the
last 4 hours of the culture period tested. Subsequently, formazan salts were
dissolved with dimethylsulphoxide (the seeded material samples were previously
transferred to a new plate) and the absorbance (A) was measured at 600 nm in an
ELISA reader. The results were normalised in terms of macroscopic area and
expressed as A⋅cm−2.


#### 2.3.2. Total Protein Content and Alkaline Phosphatase
Activity

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 μg⋅cm−2. ALP activity was
determined in cell-layer lysates (obtained by treatment of the cultures with
0.1% triton in water) and assayed by the hydrolysis of p-nitrophenyl phosphate in alkaline buffer solution, pH 10.3, and
colorimetric determination of the product (p-nitrophenol)
at 405 nm. Hydrolysis was carried out for 30 minutes at 37°C. Results were
expressed as nanomoles of p-nitrophenol produced per minute per μg of protein (nmol⋅min−1/μg Protein).


#### 2.3.3. SEM and CLSM Mcroscopy

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 (10mg⋅mL−1), in order to reduce
nonspecific staining. Cell
cytoskeleton filamentous actin (F-actin) was visualised treating the cells with
Alexa Fluor 488 Phalloidin (1:20 dilution in PBS, 1 hour) and counterstaining
with propidium iodide (1μg⋅mL−1, 10 minutes) for cell nuclei labelling. In addition,
osteoblast cell cultures were also labelled
with calcein for the visualization of calcium-containing deposits. Cultures
were incubated with calcein (25μg⋅mL−1)
for the last 3 hours of each test-point, at 37°C, washed several times in PBS,
and fixed in 3.7% paraformaldehyde (15 minutes). Labelled cultures were mounted in Vectashield
and examined with a Leica SP2 AOBS (Leica Microsystems) microscopy.


### 2.4. Statistical
Analysis

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 ofP<.05.


## 2.1. NCD-Coated Samples

NCD
coatings were produced by the HFCVD technique. Dense Si3N4 ceramics substrates were prepared
according to a processing route that can be found elsewhere [20]. Prior to
deposition, the disc-shaped samples (diameter = 10 mm, thickness = 3 mm) were
ground and polished with 15 μm diamond slurry and submitted to a 1-hour ultrasonic bath in 1 μm diamond powder suspension in n-hexane (1 g/100 mL), followed by
ultrasonic cleaning with ethanol for 10 minutes.The NCD
deposition conditions were as follows:Ts = 750°C (substrate
temperature), Tf = 2300°C (tungsten filament temperature), Ar/H2  = 0.1, CH4/H2  =  0.04, P=50 mbar (total gas pressure), F=50 mL⋅min−1 (total gas flow), td=2hours (deposition time). The HFCVD deposition
method enabled an NCD coating of 2 μm in thickness, that is, a growth rate of about 1μm⋅hr−1.
Figure 1(a) gives a representative cross-section of the
film/substrate interface as taken by scanning
electron microscopy (SEM, Hitachi S4100). The typical surface microstructure of the NCD coatings is
depicted in the atomic force micrograph
(AFM, Digital Instrument Multimode IIIa) in Figure 1(b). The diamond
nanocrystals, with an average size of 28 nm [21], are agglomerated as
micrometric clusters, leading to a root-mean-square roughness (RMS) value of 68
± 5 nm (from AFM 100×100μm scans). A previous work demonstrated that the NCD coating supported a
normal load of 400N without spalling-off and presented an interfacial crack
resistance of 6.8 Nmm−1 [22]. Before being
seeded, the NCD-coated Si3N4 samples were washed with
ethanol in an ultrasonic cleaner and sterilized by autoclaving.NCD as grown: SEM cross-sectional (a) and AFM
scan (b) views.
(a)(b)


## 2.2. Cell Cultures

### 2.2.1. Human Gingival Fibroblast Cells

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 inα-Minimal Essential Medium (α-MEM) containing 10% fetal bovine serum,  50μg⋅mL−1 ascorbic acid, 50μg⋅mL−1 gentamicin, and 2.5μg⋅mL−1 fungizone at 37°C, in a humidified atmosphere of 5% CO2 in air.  Cell outgrowth from the tissue
explants was observed 1 to 2 weeks after the beginning of the incubation. Cultures
showed a high proliferation rate and reached confluence in approximately 1 week,
with cells oriented along parallel lines, a typical feature of this culture
system [23]. Primary cultures were subcultured (0.05% trypsin, 0.25% EDTA) at
70–80% confluence.
First-passage human gingival (HG) cells were cultured (104cells⋅cm−2)
for 14 days in standard plastic culture plates and on the surface of the NCD
films. The medium was changed every 2-3 days. Cultures were characterized at 3,
7, and 14 days for cell viability/proliferation (MTT assay). In addition,
cultures were observed by confocal laser scanning microscopy (CLSM) and
scanning electron microscopy (SEM) to assess cell morphology during cell
adhesion to the substratum (first 24 hours) and throughout the culture time.


### 2.2.2. Human Bone Marrow Cells

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 of104cells⋅cm−2 in standard polystyrene culture plates and on the
surface of the NCD films. Control and seeded material samples were cultured for
21 days in the experimental conditions described above, with the culture medium
further supplemented with 10 mM β-glycerophosphate and 10 nM dexamethasone.
All the experiments were performed in the first subculture since the sequential
passage of bone marrow osteoblastic cells fallout in a progressive loss of the
osteoblast phenotype [24].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.


## 2.2.1. Human Gingival Fibroblast Cells

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 inα-Minimal Essential Medium (α-MEM) containing 10% fetal bovine serum,  50μg⋅mL−1 ascorbic acid, 50μg⋅mL−1 gentamicin, and 2.5μg⋅mL−1 fungizone at 37°C, in a humidified atmosphere of 5% CO2 in air.  Cell outgrowth from the tissue
explants was observed 1 to 2 weeks after the beginning of the incubation. Cultures
showed a high proliferation rate and reached confluence in approximately 1 week,
with cells oriented along parallel lines, a typical feature of this culture
system [23]. Primary cultures were subcultured (0.05% trypsin, 0.25% EDTA) at
70–80% confluence.
First-passage human gingival (HG) cells were cultured (104cells⋅cm−2)
for 14 days in standard plastic culture plates and on the surface of the NCD
films. The medium was changed every 2-3 days. Cultures were characterized at 3,
7, and 14 days for cell viability/proliferation (MTT assay). In addition,
cultures were observed by confocal laser scanning microscopy (CLSM) and
scanning electron microscopy (SEM) to assess cell morphology during cell
adhesion to the substratum (first 24 hours) and throughout the culture time.


## 2.2.2. Human Bone Marrow Cells

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 of104cells⋅cm−2 in standard polystyrene culture plates and on the
surface of the NCD films. Control and seeded material samples were cultured for
21 days in the experimental conditions described above, with the culture medium
further supplemented with 10 mM β-glycerophosphate and 10 nM dexamethasone.
All the experiments were performed in the first subculture since the sequential
passage of bone marrow osteoblastic cells fallout in a progressive loss of the
osteoblast phenotype [24].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.


## 2.3. Biochemical and Microscopy Assays

### 2.3.1. Cell Viability/Proliferation

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 with0.5μg⋅mL−1 of MTT for the
last 4 hours of the culture period tested. Subsequently, formazan salts were
dissolved with dimethylsulphoxide (the seeded material samples were previously
transferred to a new plate) and the absorbance (A) was measured at 600 nm in an
ELISA reader. The results were normalised in terms of macroscopic area and
expressed as A⋅cm−2.


### 2.3.2. Total Protein Content and Alkaline Phosphatase
Activity

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 μg⋅cm−2. ALP activity was
determined in cell-layer lysates (obtained by treatment of the cultures with
0.1% triton in water) and assayed by the hydrolysis of p-nitrophenyl phosphate in alkaline buffer solution, pH 10.3, and
colorimetric determination of the product (p-nitrophenol)
at 405 nm. Hydrolysis was carried out for 30 minutes at 37°C. Results were
expressed as nanomoles of p-nitrophenol produced per minute per μg of protein (nmol⋅min−1/μg Protein).


### 2.3.3. SEM and CLSM Mcroscopy

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 (10mg⋅mL−1), in order to reduce
nonspecific staining. Cell
cytoskeleton filamentous actin (F-actin) was visualised treating the cells with
Alexa Fluor 488 Phalloidin (1:20 dilution in PBS, 1 hour) and counterstaining
with propidium iodide (1μg⋅mL−1, 10 minutes) for cell nuclei labelling. In addition,
osteoblast cell cultures were also labelled
with calcein for the visualization of calcium-containing deposits. Cultures
were incubated with calcein (25μg⋅mL−1)
for the last 3 hours of each test-point, at 37°C, washed several times in PBS,
and fixed in 3.7% paraformaldehyde (15 minutes). Labelled cultures were mounted in Vectashield
and examined with a Leica SP2 AOBS (Leica Microsystems) microscopy.


## 2.3.1. Cell Viability/Proliferation

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 with0.5μg⋅mL−1 of MTT for the
last 4 hours of the culture period tested. Subsequently, formazan salts were
dissolved with dimethylsulphoxide (the seeded material samples were previously
transferred to a new plate) and the absorbance (A) was measured at 600 nm in an
ELISA reader. The results were normalised in terms of macroscopic area and
expressed as A⋅cm−2.


## 2.3.2. Total Protein Content and Alkaline Phosphatase
Activity

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 μg⋅cm−2. ALP activity was
determined in cell-layer lysates (obtained by treatment of the cultures with
0.1% triton in water) and assayed by the hydrolysis of p-nitrophenyl phosphate in alkaline buffer solution, pH 10.3, and
colorimetric determination of the product (p-nitrophenol)
at 405 nm. Hydrolysis was carried out for 30 minutes at 37°C. Results were
expressed as nanomoles of p-nitrophenol produced per minute per μg of protein (nmol⋅min−1/μg Protein).


## 2.3.3. SEM and CLSM Mcroscopy

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 (10mg⋅mL−1), in order to reduce
nonspecific staining. Cell
cytoskeleton filamentous actin (F-actin) was visualised treating the cells with
Alexa Fluor 488 Phalloidin (1:20 dilution in PBS, 1 hour) and counterstaining
with propidium iodide (1μg⋅mL−1, 10 minutes) for cell nuclei labelling. In addition,
osteoblast cell cultures were also labelled
with calcein for the visualization of calcium-containing deposits. Cultures
were incubated with calcein (25μg⋅mL−1)
for the last 3 hours of each test-point, at 37°C, washed several times in PBS,
and fixed in 3.7% paraformaldehyde (15 minutes). Labelled cultures were mounted in Vectashield
and examined with a Leica SP2 AOBS (Leica Microsystems) microscopy.


## 2.4. Statistical
Analysis

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 ofP<.05.


## 3. Results and Discussion

### 3.1. Biological Profile of the Substrate, Si3N4 Ceramic

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 [17]. Also, porous intramedullary Si3N4 rods implanted in rabbit femurs supported bone ingrowth [18]. In addition, it
was shown that a Si3N4-bioglass composite stimulated the
proliferation of MG-63 osteoblast-like cells and allowed the osteogenic
differentiation of human osteoblastic bone marrow cells, with the formation of
a mineralised matrix [19]. Considering these previous studies, in the present
work, cultures performed on standard polystyrene culture plates were used as
control in the biological assessment of the NCD coating.


### 3.2. Nonspecific
Cytotoxicity of NCD Coating

Results
regarding the behaviour of human fibroblast (HG) cells cultured on NCD coatings
are shown in Figures2 and
3, respectively, for viability/proliferation and
cell morphology throughout the 14-day culture time.Figure 2
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).
(a)(b)(c)(d)(e)(f)MTT assay
was used to assess cell viability/proliferation (Figure2), based on mitochondrial
function. Mitochondria are vulnerable targets for toxic injury because of their
crucial role in maintaining cellular function via aerobic ATP production. HG
cells showed a lag phase during the first week, followed by an exponential cell
growth. At day 3, values of MTT reduction were similar in control cultures and
seeded NCD films, suggesting an identical number of attached cells. Subsequently,
cells growing over the NCD samples presented a slightly higher proliferation
rate, but differences did not attain statistical significance.Figures3(a)–3(c)
shows the process of cell adhesion and cytoplasm spreading occurring during the
first hours of culture, with the acquisition of a progressively elongated
morphology. At 24 hours, cells showed a spindle-shaped morphology, typical of
this cell type [23], and cell-to-cell contact
(Figure 3(d)). Displaying the
typical phenotype morphology is a key requisite for the following cellular
events related to cell proliferation and functional activity [25]. Accordingly,
cells growing on the NCD coating showed a high proliferation rate, as it was
already suggested by the cell viability/proliferation results. In addition, at
long incubation time (14 days, Figures 3(d), 3(e)), cultures presented phenotype
features, namely, a flattened sheet of continuous cell multilayers displaying a
parallel alignment, which is typical of human fibroblastic cell populations
derived from adult tissues [23, 26]. The morphological events observed during
cell adhesion and spreading on NCD coating and the subsequent pattern of cell
growth described above were similar to those found in the cultures performed on
standard tissue culture plates.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 [27, 28].
Fibroblasts are ubiquitous cells on human tissues that not only function to
sustain various organs and tissues as stroma cells but also act directly to
regulate adjacent cell behaviour including migration, proliferation, and
differentiation [26]. In this way, biological evaluation with fibroblast cell
cultures might be regarded as a general bioassay, providing reliable information
concerning acute and long-term cytotoxicity or other biological responses [28].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 [23, 26], suggesting a lack
of cytotoxicity of the coating. Only few publications report on the biological
response to NCD coatings. These include the contact of blood platelet with NCD-coated
medical steel [29], the response of osteoblastic SaOs-2 cells to a composite of
NCD and amorphous carbon [30] and
also to NCD deposited by MWCVD [31], the cell adhesion of human dermal
fibroblasts on photochemically functionalized ultrananocrystalline diamond
surfaces [32], and the behaviour of several cell lines on ultrananocrystalline
diamond [33]. Most of these studies were very specific, while the present work
addresses the basal cytotoxicity profile of NCD coatings regarding ubiquitous
cells on human tissues, in agreement with the standard ISO guidelines for in
vitro cytotoxicity evaluation of medical devices [28].


### 3.3. Osteoblastic
Biocompatibility of NCD Coating

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,β-glycerophosphate and dexamethasone for 21
days, conditions that allow the expression of the entire sequence of the
osteoblastic differentiation pathway [34, 35]. Results are presented in
Figures
4–6.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.
(a)(b)(c)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).
(a)(b)(c)(d)(e)(f)(g)(h)(i)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).
(a)(b)(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 (Figure4(a)). Seeded NCD coating showed a similar pattern, but
MTT values were significantly higher at days 7, 14, and 21. Similar results
were assessed by the total protein content measurements (Figure 4(b)), which are
also an indicator of cell proliferation. HBM cells increased the production of ALP during the
first 2 weeks, attaining the maximum level by day 14, and decreasing later (Figure 4(c)). ALP is a membrane-linked polypeptide, synthesized by the
osteoblasts, that it is thought to promote crystal formation in matrix vesicles
by removing nucleation inhibitors, mediating the calcification process [36]. ALP activity, normalised as a function of the protein
content, was higher on the cultures grown on NCD surface, which may suggest
that NCD is able to stimulate osteoblast metabolic differentiated
activities.CLSM and SEM (Figures5 and
6) observations of seeded
NCD samples confirmed the favourable profile of the NCD coating suggested by
the biochemical assays. After 7–14 days (Figures
5(a), 5(b),
5(d), 5(e)), cells were well spread presenting a homogenous distribution on  the NCD surface and, by day 21, mineralised
globular structures were observed interspersed within the cell layer
(Figures 5(c), 5(f)). At low magnification
(Figure 6(a)), an elevated number of those mineralised deposits were present.
Mineralised structures were intimately associated with the fibrillar matrix
(Figure 6(b)), and the X-ray spectrum from EDS analysis (labelled with an
asterisk in Figure 6(b)) proves the inorganic nature of these structures by
showing strong Ca and P peaks. This sequence of events was similar to that
occurring on standard polystyrene culture plates (Figures 5(g)–5(i)). Quantitative
assessment of the relative abundance of mineral structures on the cell layer
was not evaluated, but SEM qualitative analysis strongly suggested that mineral
deposition density was higher on the NCD coating. This might be related with
the presence of increased cell number on the coating (as suggested by the MTT
assay), presenting an identical activity regarding matrix mineralization. Also,
induction of this phenotype feature by the NCD coating might be hypothesized,
taking in account the increased ALP activity measured on the coating.  However, the results of this study do not
provide information regarding the subjacent mechanism.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 [30], and NCD deposited by MWCVD
showed to provide good conditions for the adhesion and spreading of SaOS-2
cells as well as their viability [31].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 [37–40]. Biomolecules such as proteins, nucleic acids, lipids,
and carbohydrates possess unique properties determined by the size, folding,
and patterns at the nanoscale [41, 42]. Also, hydroxyapatite and collagen type
I, the two main components of the bone tissue, present nanofeatures [41, 42]
and, in this way, in vivo, osteoblasts interact with surfaces with a large
degree of nanometric roughness. Also, the greater surface reactivity of
nanophase materials, related to a higher number of atoms at the surface
compared to bulk, the
greater areas of surface defects and the
larger proportions of surface electron
delocalizations [43]
appear to improve cell performance. The surface
properties of nanophase materials have been shown to influence initial protein interactions that
control cell adhesion, a determinant event for the subsequent cell
proliferation and function [44, 45].
Also, the quantitative increase in the total length of particle boundaries
and total number of pores between surface particles of nanophase materials
provide a significantly extended surface area for osteoblast proliferation,
compared to micron-structured surfaces.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.


## 3.1. Biological Profile of the Substrate, Si3N4 Ceramic

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 [17]. Also, porous intramedullary Si3N4 rods implanted in rabbit femurs supported bone ingrowth [18]. In addition, it
was shown that a Si3N4-bioglass composite stimulated the
proliferation of MG-63 osteoblast-like cells and allowed the osteogenic
differentiation of human osteoblastic bone marrow cells, with the formation of
a mineralised matrix [19]. Considering these previous studies, in the present
work, cultures performed on standard polystyrene culture plates were used as
control in the biological assessment of the NCD coating.


## 3.2. Nonspecific
Cytotoxicity of NCD Coating

Results
regarding the behaviour of human fibroblast (HG) cells cultured on NCD coatings
are shown in Figures2 and
3, respectively, for viability/proliferation and
cell morphology throughout the 14-day culture time.Figure 2
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).
(a)(b)(c)(d)(e)(f)MTT assay
was used to assess cell viability/proliferation (Figure2), based on mitochondrial
function. Mitochondria are vulnerable targets for toxic injury because of their
crucial role in maintaining cellular function via aerobic ATP production. HG
cells showed a lag phase during the first week, followed by an exponential cell
growth. At day 3, values of MTT reduction were similar in control cultures and
seeded NCD films, suggesting an identical number of attached cells. Subsequently,
cells growing over the NCD samples presented a slightly higher proliferation
rate, but differences did not attain statistical significance.Figures3(a)–3(c)
shows the process of cell adhesion and cytoplasm spreading occurring during the
first hours of culture, with the acquisition of a progressively elongated
morphology. At 24 hours, cells showed a spindle-shaped morphology, typical of
this cell type [23], and cell-to-cell contact
(Figure 3(d)). Displaying the
typical phenotype morphology is a key requisite for the following cellular
events related to cell proliferation and functional activity [25]. Accordingly,
cells growing on the NCD coating showed a high proliferation rate, as it was
already suggested by the cell viability/proliferation results. In addition, at
long incubation time (14 days, Figures 3(d), 3(e)), cultures presented phenotype
features, namely, a flattened sheet of continuous cell multilayers displaying a
parallel alignment, which is typical of human fibroblastic cell populations
derived from adult tissues [23, 26]. The morphological events observed during
cell adhesion and spreading on NCD coating and the subsequent pattern of cell
growth described above were similar to those found in the cultures performed on
standard tissue culture plates.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 [27, 28].
Fibroblasts are ubiquitous cells on human tissues that not only function to
sustain various organs and tissues as stroma cells but also act directly to
regulate adjacent cell behaviour including migration, proliferation, and
differentiation [26]. In this way, biological evaluation with fibroblast cell
cultures might be regarded as a general bioassay, providing reliable information
concerning acute and long-term cytotoxicity or other biological responses [28].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 [23, 26], suggesting a lack
of cytotoxicity of the coating. Only few publications report on the biological
response to NCD coatings. These include the contact of blood platelet with NCD-coated
medical steel [29], the response of osteoblastic SaOs-2 cells to a composite of
NCD and amorphous carbon [30] and
also to NCD deposited by MWCVD [31], the cell adhesion of human dermal
fibroblasts on photochemically functionalized ultrananocrystalline diamond
surfaces [32], and the behaviour of several cell lines on ultrananocrystalline
diamond [33]. Most of these studies were very specific, while the present work
addresses the basal cytotoxicity profile of NCD coatings regarding ubiquitous
cells on human tissues, in agreement with the standard ISO guidelines for in
vitro cytotoxicity evaluation of medical devices [28].


## 3.3. Osteoblastic
Biocompatibility of NCD Coating

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,β-glycerophosphate and dexamethasone for 21
days, conditions that allow the expression of the entire sequence of the
osteoblastic differentiation pathway [34, 35]. Results are presented in
Figures
4–6.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.
(a)(b)(c)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).
(a)(b)(c)(d)(e)(f)(g)(h)(i)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).
(a)(b)(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 (Figure4(a)). Seeded NCD coating showed a similar pattern, but
MTT values were significantly higher at days 7, 14, and 21. Similar results
were assessed by the total protein content measurements (Figure 4(b)), which are
also an indicator of cell proliferation. HBM cells increased the production of ALP during the
first 2 weeks, attaining the maximum level by day 14, and decreasing later (Figure 4(c)). ALP is a membrane-linked polypeptide, synthesized by the
osteoblasts, that it is thought to promote crystal formation in matrix vesicles
by removing nucleation inhibitors, mediating the calcification process [36]. ALP activity, normalised as a function of the protein
content, was higher on the cultures grown on NCD surface, which may suggest
that NCD is able to stimulate osteoblast metabolic differentiated
activities.CLSM and SEM (Figures5 and
6) observations of seeded
NCD samples confirmed the favourable profile of the NCD coating suggested by
the biochemical assays. After 7–14 days (Figures
5(a), 5(b),
5(d), 5(e)), cells were well spread presenting a homogenous distribution on  the NCD surface and, by day 21, mineralised
globular structures were observed interspersed within the cell layer
(Figures 5(c), 5(f)). At low magnification
(Figure 6(a)), an elevated number of those mineralised deposits were present.
Mineralised structures were intimately associated with the fibrillar matrix
(Figure 6(b)), and the X-ray spectrum from EDS analysis (labelled with an
asterisk in Figure 6(b)) proves the inorganic nature of these structures by
showing strong Ca and P peaks. This sequence of events was similar to that
occurring on standard polystyrene culture plates (Figures 5(g)–5(i)). Quantitative
assessment of the relative abundance of mineral structures on the cell layer
was not evaluated, but SEM qualitative analysis strongly suggested that mineral
deposition density was higher on the NCD coating. This might be related with
the presence of increased cell number on the coating (as suggested by the MTT
assay), presenting an identical activity regarding matrix mineralization. Also,
induction of this phenotype feature by the NCD coating might be hypothesized,
taking in account the increased ALP activity measured on the coating.  However, the results of this study do not
provide information regarding the subjacent mechanism.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 [30], and NCD deposited by MWCVD
showed to provide good conditions for the adhesion and spreading of SaOS-2
cells as well as their viability [31].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 [37–40]. Biomolecules such as proteins, nucleic acids, lipids,
and carbohydrates possess unique properties determined by the size, folding,
and patterns at the nanoscale [41, 42]. Also, hydroxyapatite and collagen type
I, the two main components of the bone tissue, present nanofeatures [41, 42]
and, in this way, in vivo, osteoblasts interact with surfaces with a large
degree of nanometric roughness. Also, the greater surface reactivity of
nanophase materials, related to a higher number of atoms at the surface
compared to bulk, the
greater areas of surface defects and the
larger proportions of surface electron
delocalizations [43]
appear to improve cell performance. The surface
properties of nanophase materials have been shown to influence initial protein interactions that
control cell adhesion, a determinant event for the subsequent cell
proliferation and function [44, 45].
Also, the quantitative increase in the total length of particle boundaries
and total number of pores between surface particles of nanophase materials
provide a significantly extended surface area for osteoblast proliferation,
compared to micron-structured surfaces.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.


## 4. Conclusions

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.



---

*Source: 894352-2008-07-27.xml*