In Vitro Assessment of a Novel Boratebased Bioactive Glass

1
In Vitro Assessment of a Novel Borate-based
Bioactive Glass Roger F. Brown1, Nona L.
Adams1, Heather K. Teitelbaum2, and Richard K.
Brow2. 1Dept. of Biological Sciences and 2Dept.
of Ceramic Engineering, University of
Missouri-Rolla, Rolla, MO 65409.
Table II Alkaline Phosphatase Activity of Saos-2
Cells Cultured 5 Days on Borate Glass and 45S5
Glass Glass Alkaline Phosphatase
Activity Substrate (nmoles of pNP/30 min/500
cells) 45S5 54.8 6.4 H9 60.3 3.7
H6b 62.7 5.0 --- (control) 59.1 3.8
Values are mean standard error of mean n 4
Introduction Most of the 'bioactive' glasses
used in dental and orthopedic applications are in
the SiO2-CaO-Na2O-P2O5 system, such as 45S5 glass
developed by L. Hench and colleagues.1,2 This
in vitro investigation was conducted to assess
the biocompatibility and bioactive potential of
alkali-calcium borate-based glass as a possible
alternative for some applications. Samples of
two borate glasses, designated H6b and H9, and
control samples of 45S5 glass were incubated in a
simulated body fluid (SBF) and then analyzed for
formation of a surface layer of hydroxyapatite
(HAp), a feature essential to the bone-bonding
nature of bioactive materials.2 One test of
biocompatibility of the borate glasses involved
comparing proliferation of human Saos-2
osteoblast-like cells cultured on samples of H6b,
H9 and 45S5 glass. An addition test of
biocompatibility involved measuring alkaline
phosphatase activity, a characteristic marker of
the osteoblast phenotype, in Saos-2 cells
cultured on the borate glasses and 45S5 glass.
Results and Discussion
The X-ray diffraction patterns obtained from H6B
and 45S5 glasses soaked for 3 weeks in SBF were
virtually identical (Figure 1) and in good
agreement with the reported pattern for HAp.4
Well-crystallized HAp was not detected on H9
soaked in SBF. The formation of a HAp layer, an
essential prerequisite for bone-bonding, fulfills
one criterion for designation of the H6b borate
glass as a bioactive material.2 Saos-2 cells
exhibited a compact, rounded morphology after one
day of culturing on samples of H6b, H9 and 45S5
glass. On succeeding days of the culturing the
cells assumed a flattened, well spread
morphology, indicating they became more firmly
anchored to the glass substrates. The scanning
electron micrographs in Figure 3 reveal the
similar morphology of Saos-2 cells cultured for 4
days on H6b and 45S5 glass. Cells on both of
these glasses exhibited a similar flattened
morphology and good adherence to the substrates,
evidence the H6b borate glass is a biocompatible
material. The results illustrated in Figure 3
indicate very little proliferation of Saos-2
cells for the first 3 days of culturing on H6b,
H9 and 45S5 glasses. Large increases in Saos-2
cell density were seen at 5 days and 7 days of
culturing on the 45S5 glass samples. There was
also an increase in density of Saos-2 cells on
the H6b and H9 glass samples at 5 days and 7 days
of culturing although the increase was modest in
comparison to growth on the 45S5 glass.
The results of the alkaline phosphatase assays
presented in Table II reveal the same level of
activity of this enzyme in Saos-2 cells cultured
5 days on the H6b, H9 and 45S5 glasses.
Furthermore, the enzyme activity was very similar
to that of control cells cultured 5 days on
borosilicate glass surfaces. These results
suggest the H6b and H9 borate glasses not only
allow attachment and growth of the Saos-2 cells,
but also support continuation of bone cell
function.
Conclusions Based on these initial in vitro
results, we conclude that the H6b borate glass
forms a bioactive HAp layer upon exposure to
physiological fluid, supports bone cells growth
(albeit at a lower rate than 45S5) and permits
continuation of the osteoblastic phenotype.
Follow-up tests are planned with H6b glass
implanted in the tibia of rats to assess the
ability of this glass to bond to bone in vivo.
Experimental Reagent grade carbonates, silica,
alumina, phosphate compounds and boric acid were
mixed in the proportions given in Table I and
melted in platinum crucibles at 900-1000C.
Annealed glasses were sectioned, ground to a 800
grit finish, ultrasonically cleaned, and dry-heat
sterilized. Samples of the glasses were
incubated for 3 weeks at 37C in the SBF
formulation described by Kokubo and coworkers.3
Reacted glass samples removed from SBF were
analyzed with a Phillips XPert diffractometer.
Saos-2 cells (ATCC no. HTB 85) were cultured in
?-MEM medium supplemented with 10 fetal calf
serum, penicillin (100 U/ml), streptomycin
sulfate (100 µg/ml), plus 25 mM HEPES (pH 7.3).
Thin sections of H6b, H9 and 45S5 glass were
seeded with Saos-2 cells at a density of 20,000
cells/cm2. At intervals of 1, 3, 5, and 7 days,
Janus green was added to stain live cells and the
average numbers of stained cells per mm2 on the
glass samples were counted. Other glass sections
seeded at the same density were fixed with 2
glutaraldehyde and processed for SEM comparison
of the morphology of Saos-2 cells cultured on
H6b, H9 and 45S5 glasses. Additional glass
pieces were seeded with Saos-2 cells at a density
of 75,000 cells/cm2. After a 5 day culture
period, cells were removed from the glass pieces
by mild trypsinization, counted with a
hemocytometer, and lyzed in 0.5 Triton X-100.
Alkaline phosphatase activity in the lysates was
measured spectrophotometrically at 405 nm.
A
B
Figure 2. Scanning electron micrographs of
Saos-2 cells cultured for 4 days on thin sections
of (A) 45S5 glass and (B) H6b glass. The cells
are anchored to the substrates by multiple
lamellipodia. Grinding marks from the 800 grit
paper are visible on the surface of the glass
sections.
Acknowledgement Supported by a grant from the
Missouri Research Board.
(A)
References 1. Hench LL, Wilson J.
Surface-active biomaterials. Science 1984
226630. 2. Kim H, Miyaji F, Kokubo T.
Bioactivity of Na2O-CaO-SiO2 glasses. J Am
Ceram Soc 1995782405. 3. Kokubo T, Kushitani
H, Sakka S, Kitsugi T, Yamamuro T. Solutions
able to reproduce in vivo surface-structure
changes in bioactive glass- ceramic A-W. J
Biomed Mater Res 199024721. 4. Rehman I,
Knowles JC, Bonfield W. Analysis of in vitro
reaction layers formed on Bioglass using
thin-film X-ray diffraction and ATR-FTIR
microspectroscopy. J Biomed Mater Res 199741162.
Relative Intensity
(B)
(C)
Table I Composition of Glasses Tested Glass
Oxide Abundance (in Mole ) type
Na2O SiO2 B2O3 CaO Al2O3 P2O5 45S5 24.3 46.3
- 26.9 - 2.5 H9 5 8 40 45 1 1 H6b 15
6.5 41.5 35 1 1
20
40
25
30
35
2-theta (degrees)
Figure 1. XRD patterns of glass samples
incubated in simulated body fluid for three weeks
at 37C. Samples analyzed include (A) 45S5 (B)
H6b and (C) H9. The overlaid vertical lines
represent the powder diffraction pattern for HAp
(from JCPDS card 9-432).

Email contact rbrown_at_umr.edu
Figure 3. Histogram for comparison of
proliferation of Saos-2 cells on H6b, H9 and 45S5
glass substrates. Values are mean standard
error of mean n 4.