Journal of Biomaterials and Nanobiotechnology, 2011, 2, 163-172
doi:10.4236/jbnb.2011.22021 Published Online April 2011 (http://www.scirp.org/journal/jbnb)
Comparative In Vitro Osteoinductivity Study of
CaP Ceramics (HA, α-TCP, β-TCP) Using 10T1/2
Cells with Different Controls and Possible
Correlations with Other Systems
Juliana Tsz Yan Lee1, Kefeng Wang2, Wai Hung Tsang3,4, King Lau Chow1,3,4
1Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Hong Kong, China; 2Department of
Mechanical Engineering, The Hong Kong University of Science and Technology, Hong Kong, China; 3Department of Biology, The
Hong Kong University of Science and Technology, Hong Kong, China; 4Division of Life Science, The Hong Kong University of
Science and Technology, Hong Kong, China.
Received November 3rd, 2010; revised January 20th, 2011; accepted February 12th, 2011.
ABSTRACT
In this study, we used a pluripotent mesenchymal stem cell (MSC) model, C3H/10T1/2, to evaluate three calcium phos-
phate (CaP) materials, namely the hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate
(β-TCP). 10T1/2 cell was chosen as it has advantages over its counterparts in terms of ease of maintenance, free of
ethical concerns and also more reproducible results. ALPa enzymatic assay, RT-qPCRb, DAPIc staining and SEMd were
employed to assess the osteoinductivity of these materials. A good reference material which also acts as a scientific
controle is necessary for comparisons of results from different experimental batches and hence other materials such as
titanium, NunclonTM  plastic surface, BD FalconTM plastic surface and gold coated porous HA were also tested. The
results show that ceramics induce a more sustained osteo-differentiation state as compared with plastics. Inductivity
was found to be acting in descending order of strength with HA > β-TCP > α-TCP, which is reversed in terms of their
impact on proliferation rate (HA < TCP). This is also consistent with the results observed in SBFf study in terms of cal-
cium phosphate precipitate area coverage (HA > TCP) and in vivo osteoinductivity in terms of incidence and quality of
bone described previously (HA >  -TCP >α-TCP). These confirm the suitability of using 10T1/2 cells in cell culture
assay of osteoinductivity.
Keywords: Bone Tissue Engineering, Stem Cell, Calcium Phosphate, Hydroxyapatite, Gene Expression,
Osteoinductivity, In Vitro Test, PCR (Polymerase Chain Reaction), SEM (Scanning Electron Microscopy)
1. Introduction
Many calcium phosphate materials have been proven to
be osteoconductive, which refers to the ability of the ma-
terial to support the growth of osteoblasts [1]. In contrast,
osteoinductivity, which is the ability of a material to in-
duce undifferentiated cells into the osteo-lineage [1-3], is
an aspect that remains controversial. The osteoinductive
property of a material is usually demonstrated by bone
formation after implantation in non-osseous sites (e.g.,
subcutaneously or in intramuscular sites) [4]. It is the
most direct way of assessing osteoinductivity but in vitro
assays are highly preferred due to lower cost, shorter
experiment time and ethical issues. This requires the use
of totipotent or pluripotent cells. Adult stem cell such as
mesenchymal stem cell (MSC) model is a good candidate
due to its multipotency, relative ease of maintenance and
free of ethical concerns [5-8]. In this study, we used
C3H/10T1/2 cell as it possesses the properties of MSC
yet it is an established cell line that may provide more
reproducible results as compared to commonly used pri-
aALP: alkaline phosphatase; bRT-qPCR: reverse transcription – quanti-
tative (real time) polymerase chain reaction; cDAPI: 4',6-diamidino-2-
p
henylindole; dSEM: scanning electron microscopy; eScientific control:
The control group is practically identical to the treatment group, except
for the single variable of interest whose effect is being tested. In bioas-
says for materials on osteoinductivity, the reference material is ex-
p
ected to be inert and can also act as a reference point;f SBF: simulated
body fluid.

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
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164
mary stem cell the purity of which is difficult to control
precisely in different batches of experiments.
In in vitro assays, osteo-markers such as runt-related
transcription factor 2 (Runx2, also denoted as Cbfa 1),
alkaline phosphatase (Alp or ALPa), collagen I (Col1)
and osteocalcin (Ocn) are often monitored to evaluate the
osteoinductivity. Whether a material is osteoinductive is
determined by comparing the marker gene expression of
cells on the sample materials with that on a reference
material, ideally an inert material. Enhanced osteo-mark-
er expression is expected to be observed from an osteoin-
ductive material. Thus we also experimented on titanium
and gold coated porous HA as the reference material.
Furthermore, a suitable choice of reference material
would allow us to compare results from different batches
of experiments to a certain extent indirectly. In general,
tissue culture plastic from a given supplier has relatively
consistent surface properties and readily obtainable as
compared to materials synthesized in different batches in
small scale. Hence we compared the Alp expression of
cells on bioceramics with plastics.
On the other hand, although cell line has many advan-
tages over primary cells recovered from animals, it is not
a suitable choice unless it produces consistent conclu-
sions with in vivo study. Therefore, we analyzed our re-
sults with previous in vivo bone formation study [9]. In
order to make reliable comparisons, the materials used in
this project were synthesized in the same institute as in
the previous report and made by the same method. The
comparison with SBF study was also included for reference.
The ultimate goal of this study is to evaluate the os-
teoinductivity of calcium phosphate ceramics through
10T1/2 cell culture assays with investigation of different
control materials. The possible correlations with SBF
study and in vivo osteoinductivity were also discussed.
2. Materials and Methods
2.1. Material Synthesis
HA, α-TCP and β-TCP were synthesized by foaming
method using 5% hydrogen peroxide (H2O2) solution as
described [9]. The dried calcium phosphate cylinders
were sintered using programmed oven and then cut with
a diamond saw into circular discs of 1mm thickness and
10mm diameter. The average porosity is about 71% -
77% with macro-pores ranging from 50 to 500 m.
2.2. Material Characterization and Sterilization
The phases of different material surfaces were identi-
fied using an X-ray diffractometer (XRD) (PW1830,
Philips). Measurements were performed using a Cu-Kα
X-ray source and the XRD spectra matched with the da-
tabase using X’Pert HighScore Plus version 2.0. The
discs of materials were sterilized by autoclaving at 121˚C
for 20min and dried in a 80˚C oven. The chemical com-
position of samples after autoclave sterilization treatment
was also analyzed by an energy dispersive X-ray spec-
trometer (EDX) equipped with a scanning electron mi-
croscope (JSM-6390, JEOL, Japan). The phase of the
materials was confirmed again by X-ray diffraction
(XRD) before cell plating.
2.3. Gene Expression Study
2.3.1. Cell Su bculturing
C3H/10T1/2 cells (CCL-226™, ATCC) were cultured in
10cm culture dishes (Nunc) containing Dulbecco’s
Modified Eagle Medium (DMEM, Invitrogen) supple-
mented with 10% fetal bovine serum (FBS) and antibiot-
ics (100 U/mL penicillin and 100 g/mL streptomycin)
in a tissue culture incubator at 37˚C with 5% CO2. At
95% confluence, the cells were trypsinized, resuspended
in complete medium (DMEM with FBS and antibiotics)
and counted in a hemocytometer to evaluate the cell den-
sity. Then the cells were plated for the assays and me-
dium changed at every three days.
2.3.2. ALP Enzymatic Assay with Powder
3×104 cells/well were seeded in 96 well plates (Nunc)
with and without 1 L 2.5% (v/v) HA powder or β-TCP
powder added. After 2 days, the medium was removed
and the wells were rinsed 3 times with phosphate buffer
saline (PBS). 50 L of 0.2% Triton X-100 was added to
each well and shaken at 500 rpm for 30 min. The lysate
was mixed with 50 L 1 mM p-nitrophenyl phosphate
(Sigma) substrate solution. Absorbance at 405 nm was
measured. The absorbance of the wells without cells
plated was used as the background control. A total of
three independent assays were carried out with each as-
say performed in replicates. Calibration was performed
with p-nitrophenol (Sigma).
2.3.3. RT -qPCR
9×104 cells were seeded on each disc in 48 well plates
(Nunc). Wells without materials added (plastic surfaces)
were used as controls. RNA from the cells was extracted
by TRIzol at day 3, 6 and 12 according to the manufac-
turer’s instruction. For cells on the CaP materials, modi-
fications were made to the RNA extraction protocol as
described previously [10]. The subsequent DNase1 treat-
ment, reverse transcription and qPCR were also per-
formed as described [10]. In the qPCR experiments, a
calibrator was used for each qPCR batch so that the re-
sults from different batches can be compared.
a Alp denotes the gene that codes for alkaline phosphatase in mouse an
d
ALP denotes the alkaline phosphatase protein according to the gene
nomenclature convention ado
p
ted in the biolo
gy
field.

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
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2.4. Cell Proliferation Rate and Distribution by
DAPI Staining
9×104 cells were seeded on each disc in 48 well plates
(Nunc). At day 1 and day 3, the discs were washed with
PBS and then fixed by 4% paraformaldehyde (Sigma) for
5min at 4˚C. The permeability of cells on the discs was
increased by washing 3 times in 0.1% Triton X-100.
4',6-diamidino-2-phenylindole (DAPI) stain was applied
to the discs at 1 g/mL in 0.1% Triton X-100 for 5min,
followed by washing with Triton X-100. Fluorescence
images were taken using Olympus upright fluorescence
microscope BX41. 3 discs were used for each sample
type and 5 spots were randomly chosen from each disc. 5
to 15 images were taken for each spot due to the limita-
tion of the depth of field of the microscope and cells ly-
ing at different depths. The images were then processed
by ImageJ to obtain a clearer image and quantified using
computer software AlphaEaseFC.
2.5. Mineralisation Study by Scanning Electron
Microscope (SEM)
9×104 cells were seeded on each disc in 48 well plates
(Nunc). At day 7, the cells on the discs were washed with
PBS and then fixed by 2.5% glutaraldehyde (Sigma). The
discs were then rinsed with double distilled water and
quickly frozen by dipping into liquid nitrogen. The fro-
zen samples were transferred to punctuated microcentri-
fuge tubes and freeze dried overnight. A layer of gold of
about 20 nm thickness was sputtered on the material sur-
face with silver paint to connect the top surface to the
carbon tape. SEM images were captured at 15 kV accel-
erating voltage and 12 mm working distance using scan-
ning electron microscope (SEM) (JSM-6390, JEOL, Ja-
pan).
2.6. Statistical Analysis
P-values were calculated using two-tailed Student’s t-test
of unequal variance using Excel.
3. Results
3.1. Material Characterization
The chemical composition of the synthesized materials
was characterized by EDX and the phase by XRD. Table
1 shows the approximate quantification of the relative
element concentrations. The three major elements of the
materials (Ca, P and O) were detected and their concen-
trations are roughly equal to the theoretical values. There
is a minute amount of carbon detected on the samples
and this may be due to the adsorption of carbon dioxide
on the material surfaces. Another possibility is that car
Table 1. Quantification of elements in autoclaved HA,
α-TCP and β-TCP detected using EDX (The calculated
theoretical atomic % based on the chemical formula was
shown in the bracket).
Atomic %O Ca P C Au
HA 57 (59.1)25 (22.7)13 (13.6) 3 (0) 2 (0)
α-TCP56 (61.5)24 (23.1)15 (15.4) 3 (0) 2 (0)
β-TCP54 (61.5)25 (23.1)14 (15.4) 4 (0) 3 (0)
bonate calcium phosphates are formed. Gold was de-
tected since gold was coated on the scaffold surface to
reduce the charging effect during the measurements.
Figure 1 shows the XRD spectra of the bioceramics. The
peaks match the most intense peaks of the database. To-
gether with the EDX results, the identities of the materi-
als were confirmed.
(a)
(b)
(c)
Figure 1. XRD spectra of porous (a) HA, (b) α-TCP and (c)
β-TCP and the corresponding matches fr om the database .

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
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3.2. In Vitro Osteoinductivity Study Using
10T1/2 Cell Assay
Osteogenic development can be classified into 3 stages:
proliferation, bone differentiation and bone mineralizetion
[11]. Bone differentiation process can be used to indicate
osteoinductivity. Thus, the markers defining the bone
differentiation were monitored by both the ALP enzy-
matic assay and the RT-qPCR to evaluate the gene ex-
pression levels. In addition, when cells get more differ-
entiated, they would proliferate less and become more
mineralized. Hence proliferation was also monitored by
DAPI staining and mineralization by SEM study.
3.2.1. ALP Enzymatic Assay
This enzyme assay evaluates ALP expression at protein
level. Figure 2 shows the ALP activity of 10T1/2 cells
cultured with and without HA or
 -TCP powder. The
low ALP activity in the ‘“No cell” control’ indicates the
background ALP activity due to the adsorbed ALP from
the complete cell culture medium (with serum) was low.
The ALP activity of cells cultured with HA powder is
higher than that of cells cultured with
 -TCP powder,
suggesting the osteoinductivity of HA may be higher
than
 -TCP. At this cell plating density, the ALP activity
is lower with the addition of HA powder or
 -TCP pow-
der (comparing the ‘“No material” control’ with “Cells
with HA” and “Cells with β-TCP”). Though powder is
not normally used in bone implant applications directly,
ALP enzymatic assay using bioceremic powder may
serve as a preliminary study as HA shows higher ALP
expression in both enzymatic assay and RT-qPCR ex-
periments (to be presented in the next section) than TCP,
i.e. the results from the two studies are consistent.
3.2.2. RT -qPCR
RT-qPCR estimates Alp expression at mRNA level.
Standard Taqman® Gene Expression Assays were used
instead of self-designed primers as it would allow the
experiments to be reproduced from other researchers
more easily, which is desirable for the standardization of
assays in future.
Figure 3 shows the relative gene expression of 10T1/2
cells on different ceramics. Comparing the effect of dif-
ferent materials, there is no significant difference in ex-
pression of Cbfa1 from cells grown on different types of
materials on day 3, day 6 and day 12. The Cbfa1 expres-
sion is relatively stable across different periods. Cbfa1 is
the transcription factor regulating Ocn expression and it
is well accepted as the master switch of osteogenesis
[12,13]. The Col 1 gene is found to display an expression
level in a descending order of HA > β-TCP > α-TCP at
day 6 and there is a slight increase in expression with
time. The gene expression difference is rather small as
Figure 2. ALP activity of 10T1/2 cells cultured with and
without HA or β-TCP powder (n = 10; mean ± SEM) (*p <
0.05).
compared with Alp.
The Alp expression increases significantly with time
from day 3 for all materials under study and the increase
is larger for the calcium phosphate materials than the-
plastic control. Besides, the Alp appears to be a better
marker for comparing the osteoinductivity as contrasted
to others for monitoring the effect of different materials.
Using Alp expression as an indicator, HA is more induc-
tive to the marker expression. The effect follows the or-
der of HA > β-TCP > α-TCP, which is similar to that
observed using Col 1, although the magnitude of signal
change is greater with Alp monitored.
The Ocn expression, which is expected to increase
with time, does not show much increase during the cul-
ture period, which will be discussed in section 4.3.
3.2.3. DAPI Stai ni ng
To evaluate how different materials affect the cell prolif-
eration, the number of cells on the materials were moni-
tored using DAPI staining. DAPI is a fluorescent stain
that binds strongly to DNA and stains the nuclei of cells.
Hence it allows the cells to be detected under microscope.
Figure 4(a) shows the distribution of 10T1/2 cells which
attach to nearly the entire surface of the scaffolds made
of all three materials, HA, α-TCP and β-TCP. Each blue
dot in the photos represents a single cell. The density of
cells is higher on the cross-sections of the scaffolds than
in the middle of the pores due to the higher roughness of
these regions compared with the pore inner surfaces. It
was noted that more cells are present on the TCP discs
than on the HA discs for both day 1 and day 3.
In order to obtain the quantified result of the cell
number on the scaffolds, the number of cells on the pho-
tos was counted and the number plotted in Figure 4(b).
There is little cell proliferation for cells on HA from day
1 to day 3 while there is significant proliferation observed
in cases of using α-TCP and β-TCP as the substrates.

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
Different Controls and Possible Correlations with Other Systems
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Figure 3. Relative quantification of gene expression of Cbfa1, Alp, Col1 and Ocn in 10T1/2 cells seeded on HA, α-TCP and
β-TCP after 3, 6 and 12 days in culture. Wells in the culture plate without materials (NunclonTMΔplastic surface) were used as
reference materials. (cDNA used for Ocn quantification was double that of other genes) (n = 3 replicate discs in cell plating;
mean ± SD) (+p < 0.1, *p < 0.05, **p < 0.005).
(a) (b)
Figure 4. (a) Distribution of 10T1/2 cells on HA, α-TCP and β-TCP with DAPI staining using a 4X objective and images
processed with extended depth of field in ImageJ (scale bar = 500 m; Each blue dot represents a single cell) (b) Cell numbe rs
on HA, α-TCP and β-TCP at day 1 and 3 quantified from images with DAPI staining (n = 15 spots on three discs; mean ±
SEM) (*p< 0.05 for the proliferation rate of cells on α-TCP and β-TCP compared with HA).
These results imply that the degree of differentiation of
cells on HA is higher than that on TCP, and this notion is
consistent with the observed change of Alp expression in
these different materials.
4. Discussion
4.1. Effects of Dissolution on Gene Expression
It has been suggested that calcium and phosphate ions
would influence cell behavior and differentiation [14-21].
It is widely accepted that β-TCP can be resorbed in vivo
and we may initially expect there would be ions dis-
solved from the β-TCP, which would affect gene expres-
sion. This dissolution from HA or β-TCP was observed
in a number of studies when Ca and P free solutions were
used [22-26].
3.2.4. Morph o l og y by SEM
Figure 5 shows the morphology of 10T1/2 cells on HA,
α-TCP and β-TCP revealed by SEM. After 1 week of
culture, the cells attached and grew on the materials.
They form a layer across the pores and “nodules” impli-
cating nucleation of mineralization were observed in
some areas for all three materials, indicating that these
materials may enhance mineralization.

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
Different Controls and Possible Correlations with Other Systems
168
However, the physiological fluid is indeed supersatu-
rated with respect to the solubility of HA or β-TCP from
calculations based on methods described by Lu et al. [27].
Chen et al. [22]and Suzuki et al. [26] have also demon-
strated that the Ca concentration in the immersion me-
dium decreases with time using either SBF or cell culture
medium respectively. These observations suggest that
macroscopic dissolution of HA or β-TCP in cell culture
medium without the action of cells is negligible.
In this study, the medium is changed regularly to keep
the composition of cell culture medium as constant as
possible. A continuous flow system or a larger volume of
medium may be used in further studies to keep the
change in ion concentrations minimal.
4.2. Osteoinductivity of HA, α-TCP and β-TCP
In general, HA has higher osteoinductivity compared
with TCP based on the gene expression and proliferation
rate analysis. This indicates HA may be a better material
than TCP in inducing bone formation. On the other hand,
the cells under the condition of higher osteo marker ex-
pression have lower proliferation rate. In other words,
choosing materials which render the MSCs to have low
proliferation rate or inhibiting the proliferation may be a
possible means to achieve a higher degree of os-
teo-differentiation.
Furthermore, it is interesting to note that the Alp ex-
pression of cells on α-TCP is different from that on
β-TCP. α-TCP and β-TCP have exactly the same chem-
cal formula Ca3(PO4)2 [28-30] but differ in terms of
physical properties. α-TCP has monoclinic space groups
while β-TCP has rhombohedral space groups [28] and
α-TCP has higher solubility than β-TCP [28,30]. In min-
eralization, adenosine-5'-triphosphate (ATP) is hydro-
lyzed by nucleoside triphosphate pyrophosphohydrolase
(NTPPPH) to adenosine monophosphate (AMP) and in-
organic pyrophosphate (PPi), which gets further degraded
to yield inorganic phosphate (Pi) by alkaline phosphatase
(ALP). The inorganic phosphate thus generated forms
apatite in the presence of calcium in the extracellular
fluid [31]. The local microscopic environment (such as
pH) may be modified by cells and a lower pH would in-
crease the calcium phosphate solubility. The phosphate
concentration on α-TCP surface may be higher than
β-TCP due to its higher solubility, thus less alkaline
phosphate is required to provide the inorganic phosphate.
Besides, the cells may sense α-TCP differently from
β-TCP due to the crystal structure difference.
(a) HA at 100X (b) HA at 1 kX (c) HA at 10 kX
(d) α-TCP at 100X (e) α-TCP at 1 kX (f) α-TCP at 10 kX
(g) β-TCP at 100X (h) β-TCP at 1 kX (i) β-TCP at 10 kX
Figure 5. Morphology of 10T1/2 cells on (a-c) HA, (d-f) α-TCP and (g-i) β-TCP revealed by SEM at 100 X, 1 kX and 10 kX
magnifications at day 7 (The downward yellow arrows indicate the cells and the upward green arrows indicate the mineral-
zed structures formed). i

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
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4.3. Gene Expression of Cells on Plastic and
Effects of Osteogenic Supplement Addition
Moving a step forward to the understanding of the cho-
sen 10T1/2 cell culture assay system, the expression of
cells on other control surfaces and the effect of osteo-
genic supplements were further studied. Since the Alp
expression of cells on plastic is higher than the ceramics
at day 3, controls other than Nunclon™Δ plastics were
also tested. These include titanium, BD FalconTM plastics
and gold coated porous HA. Titanium has been success-
fully used as biomaterials [32] but without reported os-
teoinductivity. BD FalconTM culture plate has surface
chemistry different from Nunclon™Δ surface, which
may induce osteo-differentiation. Gold coating on porous
HA discs may allow the chemistry to be studied with less
consideration in the surface topography difference [33].
Figure 6 shows that cells on HA have lower Alp ex-
pression compared with all other chosen materials, which
seems to suggest HA has lower osteoinductivity com-
pared with other materials. The lower Alp expression of
cells on sample materials compared with plastics was
also observed in some results from other groups [34,35].
When we take a closer look on the Alp and Col1 expres-
sion at day 12, HA shows higher expression than plastic
even though ceramics show lower Alp expression com-
pared with plastics in short culturing period (day 3 and-
day 6). This suggests that the surface of plastic may be
better to trigger the osteo-differentiation earlier com-
pared with ceramics, but ceramics are better in sustaining
the differentiation process. It would be interesting to in-
vestigate what the surface properties of tissue culture
plastics are that render it more osteoinductive in short
culture period. Besides, it should be noted that the Alp
expression levels of cells on plastic surfaces from the two
different suppliers are different.
Figure 6. Gene expression on other materials at day 3
showing comparisons among porous HA, titanium, plastics
of different brands (Nunc and Falcon) and gold coated po-
rous HA (n = 2 PCR replicates, mean ± SD) (*p < 0.05).
On the other hand, Ocn expression has been demon-
strated to be higher when ascorbic acid and/or dexa-
methasone is added as the osteogenic supplements
[11,36]. Thus we did follow up experiments with and
without the addition of 50 g/mL L-ascorbic acid and
10-8M dexamethasone to investigate the cause of the
relative low Ocn expression. The use of osteogenic sup-
plements elevated the Alp expression on CaP materials
(Figure 7(a)), causing a larger difference among differ-
ent CaP materials and this may facilitate the quantifica-
tion. Nevertheless, the addition of osteogenic supple-
ments enhanced the Ocn expression of cells on plastics
significantly but not for cells on the ceramics (Figure
7(b)), indicating the lack of osteogenic supplements is
not the major cause of the low Ocn expression. Other
possible causes of the low Ocn expression may be the
much higher stiffness of hydroxyapatite than required by
Ocn production [37] or non-matching primers for the
Ocn transcript isoform from 10T1/2 cells due to a variety
of Ocn isoforms [38,39].
4.4. Correlation Analysis
The predictability of material bioactivity using less com-
plex systems was also assessed. Table 2 shows the sum-
mary of effects of different ceramics and by comparing
results of different systems, there are correlations among
the three systems under analysis (1. SBF study; 2. cell
culture study and also 3. in vivo bone formation). The
rationale behind the correlations was given in the sup-
plementary information. This possible correlation con-
firms that 10T1/2 cell culture can be used as an easier
and more reproducible means for studying in vitro os-
teoinductivity.
5. Conclusions
The following conclusions were drawn from this study:
(1) Among the chosen ceramics, HA shows higher os-
teoinductivity by marker gene expression analysis and
proliferation study as compared with those done with
TCPs. (2) Inhibiting the proliferation of MSCs may be a
possible means to achieve higher degree of osteo-differ-
entiation. (3) After experimenting with different control
materials, we concluded that plastic induced higher os-
teo-marker expression at the initial stage but ceramics
induced a more sustained osteo-differentiation state com-
pared with plastic. (4) Besides, the use of osteogenic
supplements may enhance Alp expression, causing a lar-
ger difference among different materials and facilitates
the quantification. (5) By comparing with the results of
SBF study and in vivo bone formation described in other
reports, the 10T1/2 cell culture system (under well co-
trolled conditions) correlateth these systems. This s wi

Comparative In Vitro Osteoinductivity Study of CaP Ceramics (HA,  -TCP,  -TCP) Using 10T1/2 Cells with
Different Controls and Possible Correlations with Other Systems
170
Table 2. Summary of results from different assay systems.
Study HA α-TCP β-TCP
Surface CaP precipitation in terms of area covered by precipitates (SBF study) [40] Whole surfacePartially covered with precipitates
Alp gene expression levels of cells on materials (ALP enzymatic assay and RT-qPCR)Strong Weak Medium
Degree of differentiation implied by proliferation study (DAPI staining) High Medium
Strong Weak Medium
In vivo bone formation in terms of incidence and quality of bone a (by Yuan & Zhang
et al. [9]) ++++ (bone) + (bone-
like tissue)
++++
(bone- like tissue)
In order to make more reliable comparisons, the materials used in this project were synthesized in the same institute as in the previous report and made by the
same method.
Figure 7. Relative quantification of gene expression of (a)
Alp and (b) Ocn relative to β-Actin in 10T1/2 cells seeded on
HA, α-TCP and β-TCP after 6 days in culture with and
without the addition of osteogenic supplements (10-8M dex-
amethasone and 50 g/mL L-ascorbic acid) (cDNA used for
Ocn quantification was double that of Alp) (n = 6 PCR rep-
licates for Alp from 2 batches of cDNA from same RNA, n =
3 PCR replicates for Ocn; mean ± SD) (*p < 0.05, **p <
0.005).
confirms the suitability of using 10T1/2 cells in cell cul-
ture inductivity study for more reproducible results.
6. Acknowledgements
We thank the constructive comments and technical as-
sistances from members of the Bioengineering lab, Prof.
King L. Chow lab, Prof. Yang Leng lab, the Materials
Characterization and Preparation Facility, the Advanced
Engineering Materials Facility of the Hong Kong Uni-
versity of Science and Technology and the National En-
gineering Research Center for Biomaterials in Sichuan
University. This work was financially supported by the
Research Project Competition launched by the Hong
Kong University of Science and Technology (Grant No.
RPC 07/08.EG02) and the Research Grants Council
(Grant no. HKUST 660407).
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