Gene-expression profiling of human osteoblasts following
treatment with the ionic products of Bioglass威
45S5 dissolution
Ioannis D. Xynos,1,2 Alasdair J. Edgar,1 Lee D.K. Buttery,2 Larry L. Hench,2,3 Julia M. Polak1,2
1
Department of Histochemistry, Hammersmith Campus, Imperial College School of Science, Technology and Medicine,
London, United Kingdom
2
Tissue Engineering Centre, Chelsea and Westminster Campus, Imperial College School of Science, Technology and
Medicine, London, United Kingdom
3
Department of Materials, South Kensington Campus, Imperial College School of Science, Technology and Medicine,
London, United Kingdom
Received 10 October 2000; accepted 19 October 2000
Abstract: The effect of the ionic products of Bioglass威 45S5
dissolution on the gene-expression profile of human osteo-
blasts was investigated by cDNA microarray analysis of
1,176 genes. Treatment with the ionic products of Bioglass威
45S5 dissolution increased the levels of 60 transcripts two-
fold or more and reduced the levels of five transcripts to
one-half or less than in control. Markedly up-regulated
genes included RCL, a c-myc responsive growth related
gene, cell cycle regulators such as G1/S specific cyclin D1,
and apoptosis regulators including calpain and defender
against cell death (DAD1). Other significantly up-regulated
genes included the cell surface receptors CD44 and integrin
␤1, and various extracellular matrix regulators including
INTRODUCTION
Bioglass威 45S5, a bioactive glass material containing
45% silicon dioxide by weight, has been shown to
stimulate osteogenesis in vitro by inducing the prolif-
eration and osteogenic differentiation of human osteo-
blasts.1 However, the molecular mechanisms that me-
diate these processes have yet to be identified. Fur-
thermore, it has not been possible to define whether
stimulation of bone synthesis by bioactive glasses oc-
curs through direct contact between substrate and
cells or through soluble ions released by these mate-
rials during their resorption. Previous studies have
Correspondence to: J.M. Polak; e-mail: julia.polak@ic.
ac.uk
Contract grant sponsor: USBiomaterials, Inc.
Contract grant sponsor: Medical Research Council UK
Contract grant sponsor: Golden Charitable Trust
Contract grant sponsor: Julia Polak Lung Transplant Fund
© 2001 John Wiley & Sons, Inc.
metalloproteinases-2 and -4 and their inhibitors TIMP-1 and
TIMP-2. The identification of differentially expressed genes
by cDNA microarray analysis has offered new insights into
the mode of action of bioactive glasses and has proven to be
an effective tool in evaluating their osteoproductive proper-
ties. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res, 55:
151–157, 2001
Key words: bioactive glass; Bioglass威; silicon; human osteo-
blasts; cDNA microarray; RCL; G1/S specific cyclin D;
CDKN1A; cyclin K; MMP-2; MMP-4; TIMP-1; TIMP-2;
DAD1; calpain; CD44; integrin ␤1
employed various molecular methods to analyse gene
expression in cells and tissues in contact with bioma-
terials and their dissolution products, including RT-
PCR,2 northern blot,3 and in situ hybridization4 analy-
ses. In general, these methods allow the investigation
of only one gene at a time. These methodologies are
therefore inadequate for conducting large-scale geno-
mic screening and developing expression profiles,
which could reveal valuable information regarding
the biological potential of biomaterials.
Current methods of identifying and comparing
global gene-expression changes include differential
display,5 subtractive hybridization,6 serial analysis of
gene expression,7 large-scale cDNA sequencing,8 ex-
pressed sequence tag analysis,9 and cDNA microar-
rays.10 Recently, we have used differential display and
subtractive hybridization to identify genes up-
regulated in two lung diseases.11,12
cDNA microarrays, however, have become the pre-
ferred method for large scale analysis of gene tran-
scription.13 The relative simplicity of the methodology
152
coupled with its high throughput capacity has given it
a clear advantage over alternative methods of analysis
of global gene expression. Using this method, expres-
sion of thousands of selected genes can be assayed
simultaneously, which facilitates monitoring differen-
tial gene expression between numerous samples in a
comparative, parallel fashion. The method relies on
the hybridization of a pool of radioactive or fluores-
cent-labeled cDNA probes synthesised from RNA to
multiple cDNA fragments immobilised onto known
locations on solid supports.
Since the development of the technology in 1995,
this methodology has been applied to a variety of
studies, including reports on the effects of different
agents on cell differentiation,14 development,15 infec-
tion,16 inflammation,17 and cancer related research.10
In this study we apply microarray methodology to
investigate the hypothesis that bioactive glasses exert,
through their dissolution products, direct control over
human osteoblast gene transcription.
MATERIALS AND METHODS
Materials
The bioactive glass conditioned medium containing the
dissolution products of Bioglass威 45S5 (a bioactive glass con-
taining 45% SiO2 w/w) was prepared by incubating 1% w/v
Bioglass威 45S5 particulate (710–300 ␮m in diameter, USBio-
materials Corp, USA) in Dulbecco’s modified Eagle’s me-
dium (DMEM) for 24 h at 37°C. Particulates were removed
by filtration through a 0.20 ␮m filter (Sartorius, UK) and the
collected medium supplemented as described above. The
elementary content of this solution in calcium (Ca), silicon
(Si), phosphorus (P), and sodium (Na) was determined by
inductively coupled plasma (ICP) analysis (ARL 3580B ICP
analyser).
Cell culture and treatments
Human osteoblasts were isolated from the trabecular bone
of femoral heads taken during total hip arthroplasty, as de-
scribed by Beresford et al.18 Average donor age was 64 ± 8
years with a male:female ratio of 1:1. Osteoblasts were
grown in DMEM supplemented with 10% fetal bovine se-
rum, 2 mM L-glutamine, 50 U/mL penicillin G, 50 ␮g/mL
streptomycin B, and 0.3 ␮g/mL amphotericin B (complete
medium) at 37°C, in 95% air humidity and 5% CO2. Osteo-
blasts were used at passages 2 and 3. For gene-expression
analysis, osteoblasts were grown onto polystyrene cell cul-
ture flasks (Nunc, Naperville, USA) at approximately 75%
confluence and then treated with bioactive glass conditioned
complete DMEM and control complete DMEM. After 48 h,
cells were released by trypsin, centrifuged, and snap frozen
in liquid N2.
XYNOS ET AL.
Extraction of RNA
Total RNA was extracted using a phenol/chloroform
method (Clontech Laboratories, Inc., Palo Alto, USA), pre-
cipitated with isopropanol and centrifuged at 15,000g at 4°C.
The RNA pellet was washed with 80% ethanol and resus-
pended in diethylpyrocarbonate-treated water. To remove
genomic DNA, the RNA samples were treated with DNAse
(0.10 U/␮L of DNase I, in DNAse I buffer). The concentra-
tion and purity of total RNA in each sample were deter-
mined by light absorbence at 260 nm and by calculating the
A260/A280 ratio, respectively. RNA integrity was confirmed
by electrophoresis on a denaturing agarose/formaldehyde/
ethidium bromide gel.
Analysis of gene expression using
cDNA microarrays
Differential gene-expression analysis of treated and un-
treated osteoblasts was performed using the ATLAS cDNA
Microarray Platform (Atlas 1.2 Human Array, Clontech
Laboratories), which allowed the simultaneous screening of
1,176 genes on paired nylon membranes. Osteoblast cultures
from four donors were compared in order to verify that the
observed differences in gene expression were not the conse-
quence of patient heterogenicity. Briefly, gene specific prim-
ers were used for the synthesis of labeled cDNA using
MMLV reverse transcriptase, which incorporated [␣32P]-
dATP (Amersham, UK). Labeled cDNA was purified from
unincorporated nucleotides by gel filtration through
Chroma Spin-200 columns (Clontech Laboratories). The in-
corporation of 32P in the probe was determined by scintilla-
tion counting. Each filter was hybridized with equal
amounts of radioactive probe. Prehybridization and hybrid-
ization was done at 68°C for 30 min and 16 h, respectively.
Arrays were washed at 68°C in low stringency buffer (2×
SSC, 1% SDS, 3 × 30 min) and then in high stringency buffer
(0.1 × SSC, 0.5% SDS, 3 × 30 min). Arrays were scanned
using a Molecular Dynamics 445 SI PhosphorImager. Nor-
malization of gene expression and data comparison was per-
formed using the AtlasImage 1.1 software (Clontech Labo-
ratories). The term “normalization” refers to using the ex-
pression levels of one or more “housekeeping genes” as
standards for measuring the expression levels of other
genes. Housekeeping genes are present in all cells. While
these genes all perform different functions and are ex-
pressed at different levels relative to one another, all exhibit
relatively constant expression levels in different tissues,
cells, developmental conditions, and diseases. When com-
paring the relative expression levels of a cDNA in treated
and control samples, gene expression was routinely normal-
ized using the expression of the housekeeping genes 40S
Ribosomal Protein S9 and 23 kDa highly basic protein.
Statistical analysis
Comparison of the elementary contents of control me-
dium and bioactive glass conditioned medium was per-
GENE-EXPRESSION PROFILING OF HUMAN OSTEOBLASTS
formed by Student’s two-tailed paired t test. A value of p <
0.05 was accepted as statistically significant. Values are ex-
pressed as mean ± standard deviation (M ± SD).
RESULTS
Inductively coupled plasma analysis
It is known that the changes in the elementary com-
position of solutions exposed to bioactive glasses are
due to an ion exchange mechanism occurring at the
material-solution interface.19 In order to characterize
the outcome of ion leaching from the material into
culture medium we analysed the elementary compo-
sition of DMEM incubated with Bioglass威 45S5 par-
ticulate and control medium in Si, Ca, P, and Na by
ICP. The concentration of Si in the bioactive glass con-
ditioned DMEM solution was increased 86,000% rela-
tive to control (16.5 ± 3.5 vs. 0.19 ± 0.03 ppm, p < 0.01).
Ca concentration was increased 10% (88.3 ± 4.6 vs. 76.3
± 1.9 ppm, p < 0.05) and P concentration was de-
creased 10% (30.4 ± 1.2 vs. 33.4 ± 0.8 ppm, p < 0.01)
relative to control, and Na concentration remained al-
most unchanged (2937 ± 49.2 vs. 2885 ± 85.4 ppm, p =
0.051).
Microarray analysis
Treatment with the ionic products of bioactive glass
dissolution for 48 h produced a diverse gene-
expression profile in human osteoblasts. Four inde-
pendent hybridization experiments comparing RNA
isolated from cells derived from different donors re-
vealed similar hybridization patterns, thus confirming
the reliability of the changes in gene expression re-
ported. One hundred ninety out of the 1,176 genes
surveyed in this study were shown to be expressed in
human osteoblasts at levels above background.
Among those, 111 genes have no previously reported
roles in osteoblast metabolism or bone homeostasis.
Additionally, our analysis revealed that 76 mRNA
species were greater than 1.5-fold up-regulated in the
treated cultures compared to the untreated control.
When a higher stringency filter was used, the analysis
identified 60 mRNA species that were up-regulated
greater than twofold in the treated cultures compared
to the untreated control (Table I). Genes up-regulated
greater than fivefold were those encoding CD44, MAP
kinase-activated protein kinase 2, integrin ␤1, and
RCL growth-related c-myc responsive gene. Repres-
sion was not as prominent as induction in our study,
with only five genes identified as down-regulated.
These included genes encoding E-16 amino acid trans-
153
porter, c-jun terminal kinase 2, polycystin precursor,
Sp2 protein, and proteasome inhibitor HPI31 subunit
(Table I).
DISCUSSION
The changes in the elementary composition of solu-
tions exposed to bioactive glasses are due to a well
characterized ion exchange mechanism occurring at
the interface of the material with these solutions.19 The
process of ion exchange is followed by network dis-
solution, which contributes further to the resorption of
these materials. ICP analysis of the bioactive glass-
conditioned medium demonstrated an 88-fold higher
Si concentration and, to a much lesser extent, changes
in Ca and P concentrations relative to control.
Although the significance of Ca in the process of
bone mineralisation is well established, the ability of
extracellular Ca to regulate cell specific responses has
only recently been demonstrated.20 Increased levels of
extracellular Ca have been shown to induce osteoblast
proliferation and chemotaxis through binding to a G-
protein coupled extracellular calcium sensing recep-
tor. The ability of osteoblasts to transport P is also
recognised as a requirement for mineralisation of
bone. The influx of P into osteoblasts via the action of
a sodium-dependent phosphate cotransporter mecha-
nism has been recently shown to trigger osteopontin
mRNA and protein synthesis and to subsequently
regulate mineralisation.21
A cellular receptor for Si has not been identified and
the precise role of Si in bone homeostasis remains un-
clear. There is evidence, however, suggesting that Si
plays an important role in bone metabolism. Solutions
of high silicon concentration induce osteoblast prolif-
eration and the expression of TGF-␤ mRNA in human
osteoblast-like cells.22 Additionally, Si administration
has been shown to increase eggshell thickness in hens
and to prevent trabecular bone loss in ovariectomised
mice,23 and a silicon-deficient diet results in impaired
collagen synthesis and defective skeletal structure in
chicken and rat.24
We have found that a solution containing Ca, P, and
Si ions can stimulate gene transcription in osteoblasts,
although at present it is unknown which elements are
responsible for the observed changes in gene tran-
scription. What is of interest is that the treatment
stimulated a specific and reproducible pattern of gene
expression by inducing a subset of genes related to
osteoblast function. Microarray experiments generate
large amounts of data. In the future, much of the raw
gene-expression data obtained using microarrays will
be deposited in searchable databases to allow public
interrogation through the internet.25
154 XYNOS ET AL.

TABLE I
List of Genes Up-Regulated or Down-Regulated Grater Than Twofold in Human Osteoblasts Treated with the Ionic
Products of Bioactive Glass Dissolution
GeneBank
Accession No. Protein/Gene Ratio Function
M59040 CD44 antigen hematopoietic form precursor 7 Cell surface receptor
U12779 MAP kinase-activated protein kinase 2 (MAPKAP kinase 2) 6 Signal transduction
X06256 Integrin beta 1; fibronectin receptor beta subunit 6 Cell surface receptor
AF040105 RCL growth-related c-myc-responsive gene 5 Growth related gene
D15057 Defender against cell death 1 (DAD-1) 4.5 Apoptosis
Y00371 Heat shock cognate 71-kDa protein 4.5 Heat shock protein
X04106 Calpain; calcium-dependent protease small subunit 4.1 Apoptosis
X59798 G1/S-specific cyclin D1 4 Cell cycle regulator
D11428 Peripheral myelin protein 22 4 Cell surface antigen
M34079 26S protease regulatory subunit 6A; TAT-binding protein 1 4 Transcription factor
D26512 Matrix metalloproteinase 14 precursor (MMP14) 3.6 Matrix component
J03075 Protein kinase C substrate 80-kDa protein heavy chain 3.5 Signal transduction
U09579 Cyclin-dependent kinase inhibitor 1 (CDKN1A) 3.4 Cell cycle regulator
L19185 Natural killer cell enhancing factor (NKEFB) 3.3 Antioxidant
M29645 Insulin-like growth factor II (IGF2) 3.2 Growth factor
L11285 Dual specificity mitogen-activated protein kinase kinase 2 3 Signal transduction
M13194 DNA excision repair protein ERCC1 3 DNA repair
U07418 MutL protein homolog 3 DNA repair
X69391 60S ribosomal protein L6 3 Transcription
M13667 Major prion protein precursor 3 Cell surface antigen
M37722 N-sam; fibroblast growth factor receptor1 precursor 3 Cell surface receptor
X79389 Glutathione S-transferase T1 3 Enzyme
L42379 Bone-derived growth factor 1 (BPGF1) 3 Growth factor
K00065 Cytosolic superoxide dismutase 1 (SOD1) 3 Enzyme
M26880 Ubiquitin 2.9 Enzyme
M17733 Thymosin beta 4; FX 2.9 Nuclear protein
J03210 Matrix metalloproteinase 2 (MMP2) 2.7 Matrix component
M37435 Macrophage-specific colony-stimulating factor (MCSF) 2.6 Growth factor
M92843 Tristetraproline 2.5 Transcription factor
D90209 cAMP-dependent transcription factor ATF-4 2.3 Transcription factor
X69550 rho GDP dissociation inhibitor 1 2.3 Signal transduction
M23619 High mobility group protein (HMG-I) 2.3 Nuclear protein
X15480 Glutathione S-transferase pi 2.3 Enzyme
X03124 Metalloproteinase inhibitor 1 precursor (TIMP1) 2.2 Matrix component
M14219 Decorin; bone proteoglycan II precursor 2.2 Matrix component
J05594 TIMP-2 2.1 Matrix component
M11233 Cathepsin D precursor 2 Enzyme
X60188 Extracellular signal-regulated kinase 1 (ERK1) 2 Signal transduction
M77234 fte-1 2 Transcription factor
AF060515 Cyclin K 2 Cell cyle regulator
M36340 ADP-ribosylation factor 1 2 Signal transduction
L35253 Mitogen-activated protein kinase p38 (MAP kinase p38) 2 Signal transduction
M36429 Guanine nucleotide-binding protein G-i/G-s/G-t beta subunit 2 2 Signal transduction
U32944 Cytoplasmic dynein light chain 1 2 Translocation
L07541 Replication factor C 38-kDa subunit 2 DNA synthesis
J00123 Proenkephalin A precursor 2 Cell surface receptor
M65212 Membrane-bound & soluble catechol-O-methyltransferase 2 Enzyme
U04847 Ini1 2 Transcription factor
L31881 Nuclear factor I (NFI) 2 Transcription factor
M30257 Vascular cell adhesion protein 1 precursor (V-CAM 1) 2 Cell adhesion
D28468 DNA-binding protein TAXREB302 2 Transcription factor
M62831 Transcription factor ETR101 2 Transcription factor
J03746 Microsomal glutathione S-transferase 12 2 Enzyme
X06985 Heme oxygenase 1 (HO1) 2 Enzyme
M35977 Vascular endothelial growth factor precursor (VEGF) 2 Growth factor
M36717 Ribonuclease/angiogenin inhibitor (RAI) 2 Nuclear protein
D16431 Hepatoma-derived growth factor (HDGF) 2 Growth factor
K03515 Neuroleukin (NLK) 2 Enzyme
M24545 Monocyte chemotactic protein 1 precursor (MCP1) 2 Cytokine
X04602 Interleukin-6 precursor (IL-6) 2 Cytokine
AF077866 E16 amino acid transporter 0.5 Transporter
L31951 c-jun N-terminal kinase 2 (JNK2) 0.5 Signal transduction
U24497 Polycystin precursor 0.5 Cell adhesion
M97190 Sp2 protein 0.5 Transcription factor
D88378 Proteasome inhibitor HPI31 subunit 0.4 Enzyme
GENE-EXPRESSION PROFILING OF HUMAN OSTEOBLASTS
Here, we will discuss those genes up-regulated that
we consider to be of particular significance. Genes
known to or likely to control and mediate proliferative
responses of osteoblasts showed distinctive patterns
of regulation. RCL, a growth promoting gene,26 was
induced fivefold. RCL is a downstream effector of the
protooncogene c-myc,27 which was moderately in-
duced 1.5-fold (unpublished data). C-myc induction
promotes cell proliferation by inducing the expression
of growth-promoting genes such as RCL.28 The ex-
pression of c-myc is regulated by the c-myc transcrip-
tional factor PuF,20 which was moderately induced
1.7-fold (unpublished data). Nuclear factor I, a tran-
scription factor that regulates osteoblast proliferation
in cooperation with the myc family of genes,29 also
was induced twofold.
Osteoblast proliferation is triggered by RCL and
other transcription factors but depends on successful
progression through the cell cycle. Cyclin D1, which
was up-regulated fourfold, is partially responsible for
the progression from the G1 resting phase of the cell
cycle to the S DNA synthesis phase by phosphorylat-
ing the product of the retinoblastoma gene,30 resulting
in the synthesis of transcription factors important for
the initiation of DNA replication. Two other cell cycle
regulators, CDKN1A31 and cyclin K,32 were also in-
duced 3.4- and twofold, respectively. These results are
in agreement with our previous findings showing that
bioactive glass induces osteoblast proliferation by
stimulating the progression of osteoblasts from the G1
phase into the S phase of the cell cycle.1,33
Interestingly, treatment by the medium containing
the ionic dissolution of bioactive glass also stimulated
the expression of the apoptosis related genes calpain34
and defender against death35 (DAD1) 4.1-fold and 4.5-
fold, respectively. Activation of the calpain system, a
proteolytic mechanism, is thought to mediate apop-
totic cell death. On the other hand, DAD1, a regulator
of N-linked glycosylation, is essential for cell survival:
DAD1 mutation was shown to induce embryonic ap-
optosis in mice.35 These findings suggest that the rate
of apoptosis is regulated by the ionic products of bio-
active glass dissolution, which is in accordance with
our previous findings that demonstrate the role of ap-
optosis as a mediator of bioactive glass induced osteo-
blast turnover and differentiation.1
Several growth factors act as local regulators of os-
teoblast function following stimulation by a variety of
extracellular stimuli. A few growth factors were in-
duced by the treatment, including insulin-like growth
factor II36 (3.2-fold increase), a key regulator of osteo-
blast homeostasis,37 and vascular endothelial growth
factor (twofold increase), a member of the fibroblast
growth factor family with osteogenic potential.38 Sev-
eral intracellular signalling molecules are involved in
osteoblast activation due to insulin-like growth factor
II and vascular endothelial growth factor, including
155
intracellular signal regulated kinase (ERK) and mito-
gen activated protein (Map) kinase pathways.37 Four
kinases were shown to be induced by the treatment,
MAPKAP kinase-2, MAP kinase-2, MAP kinase p38,
and ERK1. Also induced was the GTP binding protein,
ADP-ribosylation factor 1, suggesting that various sig-
nalling cascades may be mediating the stimulation of
human osteoblasts using the ionic products of bioac-
tive glass dissolution.
Among the cell surface receptors, highest induction
rates (sevenfold increase) were observed for CD44.
CD44 is a transmembrane glycoprotein receptor for
hyaluronic acid with cell-cell and cell-matrix adhesion
functions. Because it is expressed strongly by osteo-
cytes it is considered to be a marker of osteocytic dif-
ferentiation.39 The induction of this molecule con-
forms to our previous results suggesting that bioactive
glass induces osteoblast differentiation towards a
more mature (probably osteocytic) phenotype.1 The
integrin ␤1 type receptor was the integrin receptor
that was predominately induced due to the treatment
(sixfold increase). Integrins are heterodimeric trans-
membrane glycoproteins made of an ␣ and a ␤ sub-
unit. A wide range of integrins, particularly of the ␤1
class, is expressed by osteoblasts.40 These receptors
can recognise the Arg-Gly-Asp (RGD) peptide motif,
which is present in fibronectin and other extracellular
matrix proteins including collagen I. The role of inte-
grins in osteoblast differentiation and functional
maturation is not clear. However, some reports sug-
gest that ␤1 integrins mediate the transduction of sig-
nals from the extracellular matrix to the cells and sub-
sequently regulate osteoblast responses. 40
A primary function of osteoblasts is the production
and maintenance of extracellular matrix. Decorin is a
bone extracellular matrix component that was found
to be induced 2.2-fold by the treatment. It is a sul-
phated proteoglycan that is widely expressed during
the process of endochondral bone formation.41 The in-
duction of this molecule complements our previous
work suggesting that bioactive glass stimulates the
synthesis of sulphated proteoglycans by osteoblasts.1
Two matrix metalloproteinases, MMP-2 and MMP-14,
were up-regulated 2.7-fold and 3.6-fold, respectively.
Additionally, two of their inhibitors, TIMP-1 and
TIMP-2, were induced 2.2-fold and 2.1-fold, respec-
tively. The induction of both metalloproteases and
their inhibitors indicates an increase of extracellular
matrix turnover (for a review on the function of these
molecules in osteogenesis, see Partridge & Winches-
ter, 1996).42
In conclusion, microarray analysis of osteoblast
gene expression showed that the ionic products of bio-
active glass dissolution have a direct effect on the
gene-expression profile of human osteoblasts. One of
the most striking features of the unfolded transcrip-
tional program of human osteoblasts following treat-
156
ment with the ionic products of bioactive glass disso-
lution was the induction of genes with known roles in
processes relevant to osteoblast metabolism and bone
homeostasis. These included genes that encode prod-
ucts that can induce osteoblast proliferation (e.g.,
RCL), participate in the dynamic processes of extra-
cellular matrix remodelling (e.g., metalloproteinases),
perform differentiated functions (e.g., CD44), and pro-
mote cell-cell and cell matrix attachment (e.g., integrin
␤1). Another important feature was the coordinated
transcription of groups of genes with products that act
at different stages of a molecular process. For example,
c-myc was induced together with its transcription fac-
tor PuF and with one of its effector genes, RCL. Most
of these genes are under tight control, as indicated by
the significant induction factors. The identification of
differentially expressed genes by microarray analysis
has offered new insights into the mode of action of
bioactive glasses and has proven to be a very fast,
effective, and sensitive method for verifying their bio-
activity in vitro and identifying, in part, their biological
action.
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