Journal of Dental Research

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Bone Cell Expression on Titanium Surfaces is Altered by Sterilization Treatments

C.M. Stanford, J.C. Keller and M. Solursh
J DENT RES 1994 73: 1061
DOI: 10.1177/00220345940730050801

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J Dent Res 73(5):1061-1071, May, 1994
Bone Cell Expression on Titanium Surfaces
is Altered by Sterilization Treatments
C.M. Stanfordl,J.C. Keller', and M. Solursh2
'Dows Institute for Dental Research, College of Dentistry and 2Department of Biological Sciences, N405 Dows Institute for Dental Research,
College of Dentistry, University of Iowa, Iowa City, Iowa 52242
Abstract. Phenotypic responses of rat calvarial osteoblast-
like cells (RCOB) were evaluated on commercially pure
titanium (cpTi) surfaces when cultured at high density
(5100 cells/mm2). These surfaces were prepared to three
different clinically relevant surface preparations (1-gm,
600-grit, and 50-jm-grit sand-blast), followed by
sterilization with either ultraviolet light, ethylene oxide,
argon plasma-cleaning, or routine clinical autoclaving.
Osteocalcin and alkaline phosphatase, but not collagen
expression, were significantly affected by surface
roughness when these surfaces were altered by argon
plasma-cleaning. In general, plasma-cleaned cpTi surfaces
demonstrated an inverse relationship between surface
roughness and phenotypic markers for a bone-like
response. On a per-cell basis, levels of the bone-specific
protein, osteocalcin, and the enzymatic activity of alkaline
phosphatase were highest on the smooth 1-gm polished
surface and lowest on the roughest surfaces for the plasma-
cleaned cpTi. Detectable bone cell expression can be altered
by clinically relevant surfaces prepared by standard dental
implant preparation techniques.
Key words. Osteoblasts, Alkaline Phosphatase,
Extracellular Matrix Proteins, Dental Implants.
This paper is dedicated to the memory of Michael Solursh.
Received August 12,1993; Accepted November 23,1993
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Introduction
Favorable wound-healing responses around metallic
implants depend on critical control of the surgical and
restorative approaches used in dental implant treatment.
One critical parameter that has not been biologically
studied is the role of a clean, sterile oxide surface on an
implant. This oxide surface can alter the cellular healing
responses and potentially bone remodeling processes,
depending on the "history" of how that surface has been
milled, cleaned, and sterilized prior to placement (Kasemo
and Lausmaa, 1988). Implant surfaces altered by plasma
cleaning have been claimed to increase the initial adhesion
of cells. In turn, this has been suggested to convey a faster
healing when the surface has a high surface energy of 30
dynes/cm or greater (Carter et al., 1981; Baier et al., 1988). In
vitro, a high-energy surface will enhance the growth rate of
connective tissue cells, but without a significant effect on
the phenotypic expression of the cells (Jansen et al., 1991).
While clinical success appears to support the validity of
implant treatment over long periods of time, there is a
significant deficit in the knowledge concerning why
specific cellular events occur or do not occur on artificial
surfaces when placed in vivo. While implant failure is a
relatively rare occurrence (82% and 92% success at 10 years
for the Branemark system [Adell et al., 19901), retrospective
analysis of clinical failures often does not demonstrate an
obvious mechanism of rejection, especially when the
failures have occurred after the first year. Evaluations of the
actual amount of direct bone contact in clinically
successful cases are approximately 50% (with a range of 20-
80%) of the potential implant interfacial area (DePorter et
al., 1986; Keller et al., 1987a,b; Weinlaender et al., 1992).
Seemingly minor alterations in surface chemistry have
been hypothesized to play a role in long-term biological
1061
1062 Stanford et al.
integration (Albrektsson et al., 1986). Minor alterations in
cpTi surface oxides occur during implant preparative
procedures. For example, sterilization techniques, such as
traditional steam autoclaving, create a surface oxide that is
contaminated with various ions of N, F, Mg, Si, and Cl, as
determined on both dental implants (Klauber et al., 1990)
and on cpTi samples used in the present study (Keller et al.,
1990). To avoid contamination of the oxide surface, various
alternatives to standard autoclaving have been suggested,
including ultraviolet light (UV) (Doundoulakis, 1987;
Hartman et al., 1989), dry heat (Doundoulakis, 1987), and
plasma discharge cleaning (Baier and Meyer, 1988). These
approaches demonstrate favorable tissue reactions in vivo
(Carter et al., 1981; Baier et al., 1992).
Ultraviolet light in the higher energy ranges has a
maximal bactericidal dose at a wavelength of 254 nm,
principally through formation of nucleic acid dimerization
(Delgado and Schaaf, 1990). The use of UV irradiation for
the treatment of implants has been suggested to be an
efficacious clinical procedure (Doundoulakis, 1987;
Hartman et al., 1989), but no comparison evaluations have
been performed for cellular response to surfaces prepared
with this treatment.
Another approach to surface cleaning has been described as
micro-ashing or "plasma cleaning". In this process, a controlled
electrical gas discharge (or plasma) is used to generate an
energetic ion field that physically bombards the implant
surface (Baier and de Palma, 1970; Chapman, 1980; Kasemo and
Lausmaa, 1988). Samples are typically exposed to an ionized
low-pressure (0.1-1 mm Hg) noble gas (most commonly argon),
which removes the existing passivated oxide layer by a process
of ion stripping. Upon release of the vacuum, a new oxide
coating is spontaneously created upon reaction with ambient
oxygen (Baier and Meyer, 1988). This cleaning procedure
removes an average of 2.5 nm of the surface oxide, depending
on gas pressure, applied voltage, sample position in the
chamber, etc. (Baier et al., 1986). The resultant oxide has a
higher surface energy, as measured by contact angle
measurements (Baier and Meyer, 1988; Keller et al., 1990; Jansen
et al., 1991). Questions remain about the effects of
contamination from the walls of the ionization chamber, the
sample stand, gas purity, and conditions used upon "breaking"
the chamber seal at the end of the procedure (Kasemo and
Lausmaa, 1988). While increased cell attachment has been
described to such surfaces, no alteration in fibroblast or palatal
epithelial function occurs (Swart et al., 1992; Jansen et al., 1991).
No studies evaluating osteoblastic phenotypic responses have
been reported when these surfaces were used.
The adhesion and subsequent spreading of cells on a
biomaterial's surface are dependent on adhesion of matrix
and serum-derived proteins whose conformation and
bioactivity are, in turn, influenced by both the physical
morphology and the surface energies of that surface.
Extensive in vitro and in vivo studies have been performed
by Brunette to study the effect of surface topography on cell
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j Dent Res 73(5) 1994
adhesion, migration, and growth into implant surfaces. For
instance, when 50-nm titanium-coated surfaces were placed
percutaneously, the mesenchymal cell population oriented
only with grooves that were 22 gm or larger in depth
(Chehroudi et al., 1991). This suggested that fibroblastic cells
are capable of recognizing certain surface morphological
properties, but this study did not address any changes in
phenotypic behavior other than cell orientation. Chehroudi
et al. (1992) demonstrated, in vivo, that a grooved surface (as
small as 19 jim deep) will encourage the ingrowth of
fibroblastic connective tissue that appeared to block the
downgrowth of cutaneous epithelial cells when implants
were placed in a two-stage approach. These studies suggest
that cellular behaviors such as migration and orientation
can be influenced by surface morphology. Little has been
done to examine how alterations in differentiated cell
phenotypic behaviors can be influenced by the morphology
of an implant surface.
Using an in vitro dental implant model, this study was
undertaken to evaluate if surface roughness and differences
in surface oxide chemistry affect bone-like phenotypic
expression. Data are presented suggesting that the oxide
surface resulting from the plasma-cleaning of a cpTi
implant surface can dramatically alter the phenotypic
expression of osteoblastic cells. Further, this influence
appears to be affected by the macroscopic surface
morphology. The noncollagenous protein osteocalcin, total
collagen expression, and alkaline phosphatase activity were
chosen as bone-associated markers for this study. These
approaches allowed for an evaluation of relative
phenotypic expression as a function of the surface oxides
resulting from each treatment (Stanford and Keller, 1991).
Materials and methods
Metal sample preparation
Commercially pure titanium samples (cpTi; composition,
99.7-99.98% Ti) were prepared from sectioned bar stock (12
x 4 mm discs, lot q#F261, Alf a, Johnson Matthey) and
polished through a series of silicon carbide papers (Buehler)
to a final 600-grit prepared surface. Selected samples were
then either sand-blasted (50 gm Al oxide) or polished to a
mirror-like finish by use of a 1-jim diamond paste (Buehler).
Samples were then prepared by ultrasonic cleaning (10 min,
70% ethanol) followed by cleaning in methyl ethyl ketone
(5 min, with agitation), rinsing in purified water (Millipore
"Q" water, 15 min), then acid-passivated by exposure to 30%
HNO3 (24C) for 30 min with agitation (ASTM F86-76, 1983).
Following a 20-minute rinse with purified water, all
samples were immediately vacuum-dried and sterilized
immediately prior to use (Keller et al., 1990).
Sterilization procedures-Ultraviolet light (UV)
Samples were exposed to UV irradiation (254 nm, 300
J Dent Res 73(5) 1994 Bone Cell Expression on Titanium Surfaces
gwatts/cm2, total exposure, 1.8 x 105 gwatts, Model UVGL-
58, UVP Corp., San Gabriel, CA) for 10 min in a laminar flow
hood. Spore strips (Uvicide, Eureka, Inc., Vangard
International, Neptune, NJ) were used for efficiency
determination of this procedure. Samples were treated
immediately prior to use.
Autoclave (AC)
Samples were placed in glass Petri dishes, sealed, and treated
at 107C, 0.14 MPa, with a 20-minute pressure cycle
(American Sterilizer Company) in standard clinical
exposure conditions. Test strips (Tower Indicator Strip,
#40002, American Convertors) were used to monitor each
cycle for efficiency. To evaluate for potential contaminants
deposited on the oxide surface from this treatment, a
comparison was made with samples sterilized in an
identical manner in an autoclave with a controlled water
source (NAPCO Model 8000 DSE) in which double-distilled
water was used.
Ethylene oxide gas (ETO)
Samples were packaged in an alike manner as described
with the autoclaved samples followed by exposure to
ethylene oxide gas at 130C for 240 min at 70 MPa. Samples
were allowed to aerate for 24 h in a laminar flow hood prior
to the start of the experiments.
Plasma cleaning (PC)
Samples were positioned on glass slides in the middle of a
plasma discharge chamber (model PDC-32G Plasma
Cleaner, Harrick Scientific Corporation, Ossining, NY) and
exposed to a 100-watt discharge within a 0.07 MPa flow of
argon for 5 min immediately prior to use.
Cell culture
Primary osteoblast-like cells (RCOB) were derived from
three-day-old Sprague-Dawley rat calvaria obtained from a
standard breeding colony following the method described
by Ecarot-Charrier et al. (1983). Calvaria were isolated and
washed free of debris in Tyrode's salt solution (4C) prior to
dissection of individual parietal plates. Plates were prepared
by vigorous scraping of the endosteal and periosteal sides,
followed by a liberal removal of sutures. Central pieces of
the parietal plates were then transferred to 2 mL CMRL
1066 (Gibco) medium supplemented with 10% fetal calf
serum (Sigma, 119F-0848) and antibiotics [50 U/mL (final)
penicillin, 50 gg/mL streptomycin, and 1.25 ,ug/mL
amphotericin B] and grown for 12 days at 37'C, 5% CO2.
Medium was changed every other day. Primary outgrowths
of cells were isolated by a combination of filter-sterilized
(0.22 gm Amicon) 0.01% trypsin and 0.1% dialyzed
collagenase (Worthington) in Call and Mg++ free Saline G.
The enzyme was inactivated by transfer to medium
containing serum and the cells pelleted (900 x g, 5 min).
Packed cells were then re-suspended to a total of 5 mL,
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1063
passed through a #20 Nitex filter, and the number
determined with a hemacytometer. Trypan blue exclusion
dye studies demonstrated > 95% viability during these
experiments. Cells were plated on individual surfaces at a
density of 5 x 104/10 uL (5100 cells/mm2) for one hour prior
to being flooded with 1 mL CMRL 1066 + 10% FCS (Ahrens
et al., 1977). Identical cultures were plated on standard tissue
culture plastic ("TCP", Corning Glass Works, Corning, NY)
as a reference surface between experiments.
Osteocalcin RIA
Medium fractions from each sample were collected on the
indicated days following a complete change of the medium
for each sample. The concentration of rat-specific
osteocalcin was determined by a non-equilibrium radio-
immune precipitation assay (Chen et al., 1986; Price and
Nishimoto, 1980). Medium samples were incubated with
goat anti-rat osteocalcin polyclonal antibodies along with
pre-immune goat serum (1/40 dilution) to decrease non-
specific binding. Preliminary work demonstrated that this
assay discriminated between the rat and bovine osteocalcin
by greater than 1:10,000. Following 24 h at 4C, 0.01 iCi [1251]
rat osteocalcin was added to each tube. On the third day, an
immunoprecipitate was formed with a combination of 3%
donkey anti-goat IgG and polyethylene glycol (> 20,000
dal). The precipitate was washed two times and counted on
a gamma counter (Packard Instruments, Canberra Co.,
Downers Grove, IL) against a standard curve (0.03-3
ng/tube) of rat osteocalcin. All label and immunoreagents
were obtained from Biomedical Technologies Inc.
(Stoughton, MA 02072). Total DNA concentration per
culture was determined fluorometrically against a standard
curve of calf thymus DNA, with Hoechst 33258 used
according to the method of Brunk (1979).
Collagen expression
RCOB Collagen synthesis on biomaterial surfaces was
determined following the method of Peterkofsky and
Diegelmann (1971). Briefly, cultures were pulsed with 42
gCi/mL [L-(2,3,4,5) 3HI proline, Amersham, specific activity
= 105 Ci/mmol), starting at day three of culture for a 24-
hour period. Labeling medium was supplemented with 0.5
mg/mL (final) ascorbate and 0.6 mg/mL f-
aminopropionitrile (final). Cultures were precipitated with
20% trichloroacetic acid, followed by ethanol/ether
clarification to remove tRNA. Samples were re-suspended in
0.05 mol/L Tris-HCL, 5 mmol CaCl2, pH 7.6, and divided in
half; collagen digestion was performed upon addition of 25
mmol (final) N-ethylmaleimide and ultrapurified
collagenase (1 mg/mL Worthington CLSPA) for two hours
(37C). Prior to the assays, the collagenase preparation was
purified through an S-200 column and activity determined
against Azocoll (Sigma) by the method of Chavira et al.
(1984). Collagenous proteins (CP) were determined by
precipitation of the supernatant fraction first with ice-cold
1064 Stanford et al.
20% TCA (Count A) followed by hot (90NC) 20% TCA
precipitation for 20 min (Count B), with the remaining
pellet (considered non-collagenous proteins, NCP) dissolved
in 100% formate. Both fractions were then added to
scintillation cocktail and counted (Beckman LS 7800).
Percentage of collagen was determined with the following
formula (Sodek and Berkman, 1987):
% CP = [A/A+B+5.4 (C)]100
The 5.4 correction factor accounts for the 21% concentration
of proline and hydroxyproline in rat collagen vs. non-
collagenous proteins which usually has about 4% proline
(Peterkofsky, 1972).
Alkaline phosphatase enzyme activity
Alkaline phosphatase enzyme activity was obtained from
sonicated membrane preparations derived from bone cell
cultures grown through 12 days. This assay was based in
part on the methods of Hakeda et al. (1985) and Bessey
(1946). The cell surface was washed 2x with ice-cold Saline
G, followed by mechanical scraping with a rubber
policeman on ice. The pooled material was then pelleted
(6000 x g, 10 min, 4C) and 100 pL "lysis buffer" added [0.5%
NP-40, 0.15 mol/L NaCl, 0.05 mol/L HEPES, 0.5 mmol/L
MgCL2, and 2 mmol/L PMSF]. Following sonication of the
cell pellet (150 watts, 30 seconds total, on ice), working
solutions of para-nitrophenylphosphate (PNPP 104, Sigma,
lot:70H-50181) in 100 mmol propanediol buffer (pH 10.0)
were added, incubated for 30 min at 37C, followed by
addition of 400 pL of 0.1 mol/L NaOH to all samples and
the standard curve. A standard curve (0.5-250 nmoL) of the
product, PNP (Sigma), was prepared and incubated in
parallel with each assay. Absorption at 405 nm was
measured on an ELISA microtiter plate reader (Vmax,
Molecular Dynamics Corp.) with a linear standard curve-fit
program. Samples were normalized by fluorometrically
determined DNA/culture (Brunk et al., 1979).
Alizarin Red calcium assay
A 2% Alizarin Red dye solution (Certified Alizarin Red S, C.I.
58005, Sigma lot 60H-4402) was used in a solution-based
colorimetric assay against a standard curve of CaCl2 H20 (0.1-
21 mmol/L). Preliminary studies demonstrated a stable
absorption peak at 562 nm by 10 min (through 2 h) of color
development. This absorption maximum was stable
throughout the concentration range of free calcium used in
this series of studies. A semi-quantitative measure of bound
calcium contained within the cell and matrix fractions was
determined as follows: Individual metal samples were
jacketed with Silastic tubing pre-washed extensively in 1
mol/L EDTA. These were then sealed, the cultures washed 2x
with 1 mL PBS, and 0.5% SDS added followed by mechanical
disruption. An aliquot of the resultant matrix extract (100 ,u)
was added to 150 pL of 2% dye solution in microfuge tubes
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J Dent Res 73(5) 1994
followed by incubation (10 min, 25C). Color development
was determined in the ELISA reader, blanked against
standard solutions of the dye (in 0.5% SDS) without the added
calcium standard. Preliminary studies for this technique
indicated a reproducible (common r's = 0.994 or >)
relationship between the dye-binding assay and the mid-
range of the serial dilution used (0.1 through 21 mmol/L) with
an asymptote present at both the low and high ends of the
relationship between color development and calcium
concentration. Following a curve-fit procedure, a f our-
parameter algorithm provided the best relationship between
the experimental samples and the standard curve. This
algorithm is commonly used to account for sigmoidal-shaped
semi-log relationships in dose-response relationships and
thus is appropriate when the linear mid-range is used
(Katzung, 1984). Results of this analysis were normalized to
fluorometrically determined DNA as described above.
Cultures grown in the experimental series for alkaline
phosphatase activity and total calcium deposition (Fig. 3)
were grown in conditions also containing 10 mmol/L fS-
glycerol phosphate. Other reports evaluating the effects of
glycerol phosphate additions (Lee et al., 1992; Stanford, 1992)
demonstrated that this does not alter osteocalcin or collagen
expression markers.
Statistical analysis
Data were analyzed by an ANOVA evaluation with
Duncan's multiple-range test (SAS Institute Inc., Cary, NC).
Results
When rat calvarial osteoblast-like cells (RCOB) were placed
at relatively high density (5100 cells/mm2), we observed no
significant (p < 0.005) change in total DNA (0.186 0.061
ng/culture) over the duration of the study. In addition,
thymidine-labeled cells constituted < 6% of the culture
population over a 12-day period (Stanford, 1992).
Osteocalcin levels as a measure
of cellular response to oxide surfaces
In the modeling of potential bone-like responses to these
surfaces, the expression of osteocalcin, a specific marker of
an osteoblast-like phenotype, was selected. When
osteoblast-like cells (5100 cells/mm2) were cultured over a
12-day period, osteocalcin (OC) levels in the medium
increased gradually through day 6 but increased four-fold
between day 6 and day 8 (eg., in the case of the UV-sterilized
cpTi in Table 1). When RCOB cells were cultured on cpTi
surfaces (altered with a five-minute argon plasma
treatment), an inverse relationship was observed between
the amount of OC produced per cell and the surface
roughness (Fig. 1). Osteocalcin levels in the culture medium
were higher on the smoother surfaces (p < 0.001) in samples
obtained every other day throughout the culture period (Fig.
1). Cellular expression of this non-collagenous protein is
J Dent Res 73(5) 1994 Bone Cell Expression on Titanium Surfaces 1065

Table 1. Interval RCOB medium levels of osteocalcin from fractions collected through day 12*
(ng OC/ng DNA)
1-ptm Polished cpTi
Day UV Plasma-cleaned ETO Gas Autoclaved
4 3.2 + 0.5 4.2 + 0.4 1.9 + 0.5 2.4 + 0.2
6 7.0 + 1.3 15.5 + 1.1 8.9 + 2.1 5.1 + 0.5
8 21.1 + 3.1 34.0 + 2.8 19.1 + 3.5 15.2 + 2.3
10 25.1 + 2.0 39.9 + 0.2 20.2 + 0.1 16.1 + 0.3
12 26.8 + 2.2 49.8 + 4.0 21.2 + 2.5 19.8 + 1.3

600-grit cpTi
4 3.1 + 0.5 4.0 + 0.6 3.5 + 0.6 2.7 + 0.5
6 8.9 + 0.1 4.7 + 1.9 4.4 + 0.7 3.6 + 1.0
8 23.9 + 1.7 8.3 + 1.6 18.4 + 2.2 12.7 + 1.6
10 38.3 + 1.3 18.4 + 3.2 28.2 + 0.1 19.7 + 1.7
12 37.4 + 3.2 29.8 + 0.8 31.4 + 1.7 28.6 + 0.4

50-jm Sand-blasted cpTi

4 0.4 + 0.1 0.4 + 0.1 0.4+ 0.1 0.4 + 0.1
6 1.2 + 0.1 0.4 + 0.1 0.5 + 0.1 1.2 + 0.2
8 36.4 + 1.3 7.5 + 0.7 17.6 + 0.0 23.5 + 0.5
10 22.0 + 7.9 6.3 + 1.0 28.8 + 12.5 17.4 + 7.7
12 25.2 + 0.1 9.3 + 1.5 11.1 + 1.1 11.8 + 1.1
* Measured with a Goat anti-rat RIA with medium fractions from each collection day. n = 3 replicates/group/time point; mean and
standard error of three trials.

quite evident when total levels of expression are evaluated
and compared with the other sterilization treatments (Table
2 and Fig. 2). As a reference for comparisons, osteocalcin
expression was evaluated from RCOB cultures on tissue
culture plastic (TCP) and found to be equal to the 600-grit
cpTi surface (Table 2 and Fig. 2). Since osteocalcin can bind
to mineralized matrix (Hauschka et al., 1989), cell matrix
levels of OC were determined but found never to represent
greater than 3% relative to the medium levels under
conditions where mineralization was not accentuated by
the addition of exogenous [-glycerol phosphate. When
cumulative osteocalcin expression from cultures on all the
surfaces is compared (Fig. 2), the effect(s) of surface
roughness are illustrated by a significantly (p < 0.001)
greater level of expression on the PC-smooth titanium
surface relative to the smooth plastic reference surface.
Similar values were obtained for cultures grown on plastic
vs. the 600-grit surface while a significant (p < 0.001) 60%
drop in expression on the sand-blasted metal was described
over the entire culture period. In contrast, OC expression on
sand-blasted UV-, AC-, and ETO-treated surfaces was
higher than on the PC surface (Table 2 and Fig. 2). In
general, RCOB cells grown on AC-treated cpTi
demonstrated the most consistent expression of osteocalcin
of the three surface roughness (Table 1 and Fig. 2).
In order to evaluate whether surface contamination was
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imparted on the AC samples through use of the water
source in the standard clinical autoclave, a comparison
experiment was run with 600-grit cpTi samples (N = 4)
sterilized in a standardized autoclave in which ultrapure
(doubly-distilled) water was used. Comparison values for
osteocalcin expression demonstrated trends identical to
those observed in the present study, without a statistical
difference (p < 0.05) between the two AC treatments.
Collagen expression on cpTi prepared
to two levels of surface roughness
Collagen expression by bone-like cells is a crucial
component of the mineralizing extracellular matrix. It was
therefore of interest to evaluate if sterilization treatments
would demonstrate similar effects on collagen expression
on the moderately roughened 600-grit dental-implant-like
surface and the highly polished surface. Total collagen
expression (as determined by the sensitivity to ultrapurified
collagenase) did not vary significantly (p = 0.05) among the
four sterilization treatments. Collagen expression tended to
be greater on cpTi than on the TCP reference surface (Fig. 3).
Alkaline phosphatase activity
and total calcium accumulation
Phenotypic characteristics of bone cells in vitro are often
characterized by relatively high alkaline phosphatase
1066 Stanford et al.
60
< 50
z z
0 0
- 1,um
C]CD 40
600 grit
0 100
30
CU
m
0 /SB
0
z.' 20
co0
0)
C- 10
0 PC
6 8 10 12
Time (days)
Figure 1. Medium levels of osteocalcin derived from RCOB cells
grown on plasma-cleaned commercially pure titanium (cpTi).
Surfaces were prepared to one of three roughness values: 1-gm or
600-grit polish or sand-blasted (SB). Medium fractions were
collected every other day. n = 3 replicates/group/time point; mean
and standard error of three trials.
enzyme activities. Therefore, enzyme activity was evaluated
from RCOB cultures allowed to grow on the 600-grit cpTi
surface following sterilization with UV or PC. These two
treatments were selected because they resulted in the
greatest difference in osteocalcin expression with surface
morphology (PC) or allowed for the greatest average level of
osteocalcin expression independent of the surface
roughness (UV). Alkaline phosphatase activity tended to
follow a trend of elevated activity on the smoother surfaces
than on the sand-blasted surface. For the highly polished
titanium surface exposed to UV irradiation, a gradual rise in
activity through day 8 was described, with a rapid increase
in AP activity by day 10 (Fig. 4). Again, the elevation of a
phenotypic marker, in this case a functional enzyme
activity (vs. protein levels), was greater on the smoother
surfaces (Fig. 4 through day 10 and Fig. 5). This trend was
more consistent with the PC-treated surfaces after day 10 in
culture. When RCOB cells were cultured on the plasma-
cleaned surfaces, AP activity was greater on the smoother
surfaces following day 10 or later (Fig. 5). Interestingly,
under either sterilization treatment, the level of enzyme
activity on the rougher sand-blasted surfaces (as a function
of the number of cells present) was always significantly
lower (p < 0.001, Figs. 4 and 5). In order to evaluate if the
differences in AP activity may have an effect on
mineralization, total calcium levels were measured by an
Alizarin Red colorimetric assay (Fig. 6). Calcium levels were
normalized to the number of cells present at each time
point. Total calcium levels remained at a steady level
through the first six days of culture (p < 0.05, with 93.68 jg
calcium/ng DNA for UV-treated surfaces vs. an average of
65.66 ,ug calcium/ng DNA for the PC-treated surfaces).
Following day eight of the study, RCOB cultures on the UV-
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J Dent Res 73(5) 1994
200
180
160
- TCP
140
0)
a
- 120 1 pm cpTi
.0-
600 grit
0
0 80
0
0 60
[::: SB
0)
C3 40
20
0
UV AC ETO
Sterilization Treatment
Figure 2. Total osteocalcin levels as a function of sterilization
treatment and surface roughness. Accumulated release of
osteocalcin from medium fractions over a 12-day period
demonstrated that expression varied on the three surface
roughnesses with sterilization treatment as indicated. Surfaces were
prepared to one of three roughness values: 1-gm or 600-grit polish or
sand-blasted (SB). TCP = tissue culture plastic; UV = ultraviolet-light-
sterilized; PC = plasma-treated; ETO = ethylene oxide; and AC =
autoclave-sterilized cpTi. n = 15/surface roughness/treatment/trial;
mean and standard error of three trials.
treated surfaces demonstrated higher (p < 0.05) levels of
total calcium deposition vs. the PC-treated surfaces at any of
the remaining time periods evaluated (Fig. 6). Total calcium
levels were observed to rise to a maximum of 277 jg
calcium/ng DNA by day 12, in contrast to the PC-treated
group, which had levels of 187 ,ug calcium/ng DNA. In
addition, total calcium levels rose faster on the UV-treated
surfaces (starting at day eight) vs. a two-day lag for the PC-
treated surfaces before a significant increase occurred in
total calcium levels.
Discussion
Rat calvarial osteoblast-like cells isolated from primary
outgrowths are a biologically responsive system, highly
useful for biocompatibility assays. High-density culture
conditions have previously been implicated in promoting a
differentiated phenotype in chicken (Gerstenfeld et al.,
1987) and rat calvarial cultures (Chen et al., 1986), along
with a number of cell lines (Folkman and Moscona, 1978).
These results suggest that a minimal cell density (leading to
alterations in cell shape and cytoskeleton) may be needed
before differentiation can proceed (Folkman and Moscona,
1978; Osdoby and Caplan, 1979; Gerstenfeld et al., 1987). The
use of enriched osteoblastic cultures from rat calvaria for
evaluation of responses to metabolic stimuli, cytokines, and
auto/paracrine growth factors has been reported (Ecarot-
Charrier, 1983; Bellows et al., 1986). When osteoblastic cells
are derived from primary outgrowths, an enriched
j Dent Res 73(5) 1994 Bone Cell Expression on Titanium Surfaces 1067

Table 2. ANOVA for osteocalcin levels -i 50

Source df ss Mean Square

2 40

Between 33 90501 8227.4

- TCP
Within 288 57838 602.5 co 30
C
(D 1 pm cpTi
Total 321 88171 C,)
F = 13.656; P < 0.0001 20 600grit cpTi

Duncan's Multiple Comparison Test: Groups connected were not 0)

0 10
significantly different (p > 0.05), while non-connected groups had a
p value < 0.001. ~ 0
TCP UV PC ETO AC
Roughness/Sterilization Sterilization Treatment

lI.tm/PC Figure 3. Effects of surface roughness and sterilization treatment on

600 g/UV
1 .tm/UV
Il m/ETO
1 , m/AC
600 g/PC
600 g/ETO
600 g/AC
SB/UV
SB/ETO
SB/AC
TCP
SB/PC
population of cells is obtained that is positive for the
expression of type I collagen, exhibits high levels of alkaline
phosphatase activity, and demonstrates expression of non-
collagenous bone proteins (Stanford et al., 1992). In
evaluating the expression of osteoblastic cells used in this
study, it was first determined that proliferation occurred
only in a minor (< 5%) proportion of the heterogeneous cell
population. Therefore, cellular responses-as measured by
osteocalcin release, collagen expression, and alkaline
phosphatase activity in response to the implant-like
surfaces-were indicative of phenotypic responses of the
cultures without a significant change in the number of cells
over the assay period. Moreover, the evaluation of these
markers was normalized to the amount of DNA
fluorometrically determined in each trial to account for any
variation in cell numbers between experiments.
Titanium will spontaneously form a surface oxide that
varies both in thickness and composition when evaluated
with standard physical/chemical methods. Previous work
has demonstrated that differences in these parameters will
reflect variations in preparation and sterilization history
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percent collagen expression. Cultures were labeled with 42 gCi/mL
[3H1-proline (SA = 105 Ci/mmol) in the final 24 h of incubation,
starting at day 3. Percentage of collagenase-sensitive material
determined with an n = 4/surface roughness/treatment/trial; means
and standard deviations of two trials.
(Kasemo and Lausmaa, 1988; Keller et al., 1990). In general,
the oxide surface resulting from standard implant
preparation techniques is highly irregular both in thickness
and composition (Ducheyne, 1988; Keller et al., 1990).
Therefore, acid-passivation treatments were used as a
method of standardizing the surface oxide prior to each
sterilization treatment used in this study (ASTM standard
F86-76, 1983). Acid passivation of the metal surface did not
appear to alter the surface macro-morphology but imparted
a standardized oxide thickness and composition, ideal for
this in vitro study (Keller et al., 1990). Previous work by
Keller et al. (1990) demonstrated autoclaved and ethylene-
oxide-sterilized 600-grit cpTi surfaces exhibiting an
increase in oxide thickness from 3 nm (on the passivated
reference sample) to 5 nm for ETO-treated cpTi and 25 nm
for steam-autoclaved samples, by Auger spectroscopy. Upon
chemical analysis, these authors found that the autoclaved
surfaces were contaminated with various ion species (e.g.,
Fe, Cl, N). This agreed with previous work by Doundoulakis
(1987) and Baier et al., (1982), suggesting that autoclave
treatment of surfaces resulted in the deposition of
contaminating films that result in a lower surface energy.
These films appear to alter the surface properties of the
substrate by increasing the surface contact angle and
leading to the suggestion that lower energy surfaces will
alter the ability of cells to attach. Glow-discharge or
plasma-cleaned cpTi surfaces, on the other hand,
demonstrated increased RCOB cell adhesion with plasma
exposures up to 5 min (the time used in the present study),
but adhesion is diminished with longer exposures (Jansen et
al., 1991; Swart et al., 1992). This method of surface cleaning
has been promoted to increase cell attachment through
formation of an elevated surface free energy, leading to the
1068 Stanford et al.
1.00
z 0.80
a 0
0)
c
- a
0.60
E 600 grit
a-
z 0.40
a.
0 0
0 0
E 0.20
0.00
6 8 10 12
Time (days)
Figure 4. Temporal increases in alkaline phosphatase activity in
membrane fractions derived from RCOB cultures grown on
ultraviolet-light-irradiated c pTi as a function of surface roughness.
Samples were prepared as described in "Materials and methods".
Surfaces were prepared to one of three roughness values: 1-gm or
600-grit polish or sand-blasted (SB). n = 6 replicates/group/time
point/trial; mean and standard error of two trials.
unproven implication of increasing biological acceptance
(Van der Valk et al., 1983; Schakenraad et al., 1986; Baier et
al., 1992). To date, few studies have attempted to study how
alterations of solid-state surface energy and composition
will alter the phenotypic expression of bone-like cells in a
controlled environment. On the other hand, in vivo
approaches have been used to questions concerning the
effects of plasma cleaning. For instance, Baier et al. (1992)
recently reported that plasma-cleaned implants increased
pull-out strengths in a rabbit femur model with adherent
calcified material, suggesting a cohesive mode of fracture.
Variables such as surface morphology and failure loads
were not reported, and the control or comparison groups
were unclear. Jansen et al. (1991) showed in vitro that
plasma-cleaning titanium increased dermal fibroblast
adhesion but did not alter growth kinetics or cytoskeletal
morphology. Therefore, it was of interest to determine if
bone-like phenotypic markers could be altered by exposure
to titanium prepared with these different clinically relevant
surface preparations.
The results of this study suggest that the plasma-
cleaning treatment may have an influence on phenotypic
RCOB cell behavior in addition to the elevation in cell
adhesion previously described (Swart et al., 1992). When
osteoblast-like cells were plated onto cpTi prepared to three
widely varying surface morphologies, two markers of a
bone-like phenotype, osteocalcin and alkaline phosphatase,
demonstrated significant differences between surface
morphology and alterations in the oxide composition
resulting from the sterilization treatment. When titanium
surfaces were prepared to three roughness values, followed
by a five-minute plasma glow discharge, the highest level of
OC expression and AP activity was described on the
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j Dent Res 73(5) 1994
1.00
z 0.80
1,Pm C] - 1pm
1-
0.60
.Ea 600 grit
/ SB W SB
z 0.40
FE 0.20
0.00
6 8 10 12
Time (days)
Figure 5. Temporal increases in alkaline phosphatase activity in
membrane fractions derived from RCOB cultures grown on plasma-
cleaned cpTi as a function of surface roughness. Samples were
prepared as described in "Materials and methods". Surfaces were
prepared to one of three roughness values: 1-gm or 600-grit polish or
sand-blasted (SB). n = 6 replicates/group/time point/trial; mean and
standard error of two trials.
smoothest high-energy surface. In comparison with cultures
grown on the sand-blasted surface, the 1-gm polished
surface manifested a five-fold-greater (Fig. 1) level of OC
expression as a function of the population of cells present.
Titanium surfaces exposed to UV, gas, or autoclave
treatments did not demonstrate a similar relationship
between sterilization and surface roughness (Table 1, Fig. 2).
In contrast, when collagen expression was evaluated from
cultures grown on identical surfaces, no differences could
be discerned. The technique used for evaluation of collagen
provided a general measure of collagen production via
enzymatic digestion of radiolabeled material but does not
distinguish collagen types. As such, it is possible that
collagen expression may vary among the various surfaces.
The clinical significance of phenotypic markers may be
more associated with alterations of specific bone proteins,
since they are altered to a greater degree by surface histories
than by general markers such as the quantity or type of
collagen produced on an implant surface. In previous work,
Stanford et al. (1992) showed that RCOB cultures express a
matrix rich in predominantly type I, with some type III
collagen. Type II collagen was not detected either
histochemically or biochemically. These results agree, in
part, with the findings of Puleo et al. (1991), where RCOB
collagen expression was not found to vary among a range of
widely different metal surfaces.
Conclusions based on these observations must be
carefully drawn, because the actual biological role of the
osteocalcin protein is still unclear. It is interesting that
osteocalcin has been suggested to mediate a role in bone
resorption, since warfarin-treated bone segments (being
void of post-translationally modified osteocalcin) are not
resorbed by osteoclast-like cells, in vitro or in vivo
j Dent Res 73(5) 1994 Bone Cell Expression on Titanium Surfaces
400
z
0
300
C]
E UV-cpTi
.0 200
PC-cpTi
I-
0 widely characterized integrin systems), with the net effect
0)
100
c
nL
0
2 4 6 8 10 12
Time (days)
Figure 6. Temporal accumulation of total calcium in RCOB
micromass cultures on cpTi as a function of surface roughness
following UV or plasma cleaning. Total calcium was measured by
an Alizarin Red S colorimetric assay as described in "Materials and
methods". n = 6/group/time point, mean and standard deviation,
representative of two trials.
(Glowacki and Lian, 1987; Glowacki et al., 1991; DeFranco et
al., 1991). The results of the present study suggest that
alterations of osteocalcin composition within the matrix
surrounding an implant may be influenced by implant
surface conditions.
Quantifying the total content of calcium present in the
RCOB cultures is one approach to evaluating differences in
biological responses, since this measure represents the result
of multiple cellular and extracellular processes
(Zimmermann et al., 1991; Groessner-Schreiber and Tuan,
1992). In this study, a colorimetric approach to quantifying
total calcium by a non-radioactive approach has been
presented. Alizarin Red is a common histological stain but
can be used as a solution phase measure of total calcium
following extraction of the cell layer. When total calcium
was normalized to DNA concentration, a relative measure of
mineralization per cell is created (Fig. 6). Mineralization in
this culture system has previously been demonstrated to
depend upon alkaline phosphatase activity for the
initiation, but not the progression, of mineral formation
(Bellows et al., 1986, 1991; Stanford, 1992). Given that AP
activity is needed for initiation of mineralization, the
decrease in AP activity described in this study for the
rougher surfaces (Figs. 4 and 5) follows with an identical
trend in total calcium levels (Fig. 6). This depression in
activity may not be related to final biomechanical
properties (such as pull-out strengths), because enzyme
activity occurs prior to osseous wound-healing. The final
biomechanical strength is derived from both the type and
degree of micro- and macro-retentive features of an implant
surface (Brunski, 1992). In this study, though, a reproducible
high-density micro-environmental system has been created
on the test surface, followed by evaluation of individual
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1069
cellular responses. Subtle differences in cellular expression
(probably induced through alterations in the cytoskeleton)
can reflect the cell's ability to respond to matrix proteins
that are believed to coat the biomaterial surface. Cellular
responses will represent the effect of protein adsorption
(composition, quantity, and, most probably, conformation)
and the state of a cell membrane's adhesive systems (eg., the
being an alteration in cell shape. It is probably these
alterations in cell shape that induce the changes in protein
expression and the enzyme activity described in this study.
Acknowledgments
This investigation was supported by USPHS Research
Grants DE00234, DE08540, and KD16DEO0175 from the
National Institute of Dental Research, National Institutes of
Health, Bethesda, MD 20892.
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