Supplementary Data
S.1 Powder X-ray Diffraction
The crystallinity and particle size of β-TCP micro- and nanoparticles were analyzed by
means of an Empyrean X-ray diffractometer (Panalytical). Each sample was analyzed to verify
the compound purity and compare the structure of micro- and nanoparticles.
Figure S1 shows the X-ray diffraction patterns of β-TCP micro- and nanoparticles. Both
samples were verified using Data Collector (4.0a). As signified by the minor peak shifts and
differences in signal heights and widths, the crystallinity and particle size of the microparticles
differ from that of the nanoparticles.
Figure. S1 XRD analysis of β-TCP micro- and nanoparticles
S.2 β-TCP Particle Cytotoxicity in Suspension
The effect of β-TCP particles on the proliferation of stem cells was evaluated after
treating cells with varying concentrations of β-TCP particles over a controlled time. Cells were
seeded at 2.5 x 104 cells/sample (n=3) and incubated for 24 hours to allow for proper adhesion.
After initial 24-hour incubation, media was removed and varying concentrations (wt/vol%) of βTCP, both micro- and nanoparticles, (2.5%, 2.0%, 1.5%, 1%, 0.5% 0.1% and 0.01%) were
exposed to hASCs for 24 hours (acute response), 3 days, and 7 days. Hank’s balanced salt
solution (90% of media volume) and alamarBlue® reagent (10% of media volume) were added
to each well and incubated at 37 °C and 5% CO2 for 2 hours. The hASCs live control was
cultured on tissue-treated polystyrene with no exposure to β-TCP particles, and the dead control
was hASCs exposed to 70% ethanol. The fluorescence was measured at an excitation wavelength
of 530 nm and an emission wavelength of 595 nm using a fluorescence plate reader.
hASCs viability was determined using metabolic activity assay after exposure to nanoand micro-sized β-TCP particles at varying concentrations: 0.01%, 0.1%, 0.5%, 1.0%, and 2%
(%w/v). Viability was verified by measuring the metabolic activity of hASCs after 1, 3, and 7
days of particle exposure (Figure S2 A-C). alamarBlue® reagent is a colorimetric/fluorometric
indicator of cellular metabolism and is often used as a viability indicator by measuring the
reduction of the active reagent within living cells. Figure S2 (A-C) shows the cellular activity of
hASCs treated with varying β-TCP particle solutions, both for nano- and micro-sized particles
over a 7-day study with cellular activity measured at 1, 3, and 7 days; hASCs cultured without
exposure to β-TCP particles were used as a positive control.
Exposure to particles for one day (Figure S2A) revealed that micro-sized β-TCP particles
have little cytotoxic effect on hASCs, regardless of concentration, whereas cellular metabolic
activity decreased with increasing nanoparticle concentration. Percent viability of 1 day
nanoparticle-treated (wt%) cells was 86.3% (0.01%), 83.7% (0.1%), 96.9% (0.5%), 75.4% (1%),
44.4% (1.5%), 25.3% (2%), and 18.5% (2.5%); microparticle viability was 96.2% (0.01%),
106.5% (0.1%), 119.1% (0.5%), 106.0% (1.0%), 99.0% (1.5%), 126.8% (2.0%), and 107.5%
(2.5%). Exposure to nanoparticles for 3 days (Figure 2B) revealed significant (P < 0.05)
cytotoxicity, compared to the control, at all concentrations above 1%, while exposure to microsized particles showed minimal cytotoxicity. At day 3, viability of nanoparticle-treated cells was
133% (0.01%), 132% (0.1%), 112% (0.5%), 88% (1.0%), and essentially zero metabolic activity
with concentrations above 1%. The average viability after microparticle exposure for 3 days was
113% (0.01%), 111% (0.1%), 111% (0.5%), 101% (1.0%), 58% (1.5%), 104% (2.0%), and 70%
(2.5%). Exposure to particles for 7 days (Figure 2C) revealed the greatest impact on hASCs
viability for each particle size and concentration. The 7-day viability results of nanoparticletreated cells were 59.6% (0.01%), 37.5% (0.1%), 31.1% (0.5%), 20.9% (1.0%), 0.06% (1.5%),
1.0% (2.0%), and 0.64% (2.5%). The viability results of microparticle-treated cells were 62.0%
(0.01%), 13.3% (0.1%), 19.9% (0.5%), 35.3% (1.0%), 27.6% (1.5%), 3.3% (2.0%), and 25.2%
(2.5%). In general, nanoparticles were found to be more toxic than microparticles at most time
points and concentrations.
Figure S2. β-TCP micro- and nanoparticle cytotoxicity in suspension at 0.01, 0.1, 0.5, 1,
1.5, 2, and 2.5 (w/v) % at A) 24 hours, B) 3 days, and C) 7 days
IL-6 represents a common inflammatory interleukin. qPCR was performed to quantify
IL-6 expression over 7 days. Figure S3 shows the IL-6 (-ΔΔCt) values over a 7-day exposure to
nano- and microparticle solutions. Particle size and dosage did not have significant impacts on
IL-6 expression. IL-6 expression after 7 days of exposure was significantly less (P < 0.05) than
IL-6 expression after 1 day of exposure.
Figure S3. IL-6 expression (ΔΔCt) of hASCs at A) 24 hours, B) 3 days, and C) 7 days
exposure to 0.01, 0.1, 0.5, 1, 1.5, 2, and 2.5 (w/v) % nano and micro β-TCP particle solutions