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Mechanical Properties and Antibiotic Release Characteristics of Poly(methyl
methacrylate)-based Bone Cement Formulated with Mesoporous Silica Nanoparticles
Kumaran Letchmanana,*
, Shou-Cang Shena, Wai Kiong Ng
a, Poddar Kingshuk
b, Zhilong Shi
b,
Wilson Wangb, Reginald B.H. Tan
a,c,*
a Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology
and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore
b National University of Singapore, Department of Orthopaedic Surgery, 1E Kent Ridge
Road, NUHS Tower Block Level 11, Singapore 119228, Singapore.
c Department of Chemical and Biomolecular Engineering, National University of Singapore,
4 Engineering Drive 4, Singapore 117576, Singapore
Corresponding author. Tel.: +(65) 67963880
Corresponding author. Tel.: +(65) 67963841
ABSTRACT
The influence of mesoporous silica nanoparticles (MSNs) loaded with antibiotics on the
mechanical properties of functional poly(methyl methacrylate)-(PMMA) based bone cements
is investigated. The incorporation of MSNs to the bone cements (8.15 wt%) shows no
detrimental effects on the biomechanical properties of the freshly solidified bone cements.
Importantly, there are no significant changes in the compression strength and bending
modulus up to 6 months of aging in PBS buffer solution. The preserved mechanical
properties of MSN-functionalized bone cements is attributed to the unchanged
microstructures of the cements, as more than 96% of MSNs remains in the bone cement
matrix to support the cement structures after 6 months of aging. In addition, the MSN-
functionalized bone cements are able to increase the drug release of gentamicin (GTMC)
significantly as compared with commercially available antibiotic-loaded bone cements. It can
be attributed to the loaded nano-sized MSNs with uniform pore channels which build up an
effective nano-network path enable the diffusion and extended release of GTMC. The
combination of excellent mechanical properties and sustainable drug delivery efficiency
demonstrates the potential applicability of MSN-functionalized PMMA bone cements for
orthopedic surgery to prevent post-surgery infection.
Graphical Abstract
Key word
Antibiotics, biomechanical properties, PMMA bone cement, sustained release, compression
strength, bending modulus.
1. Introduction
Post-operative implant-associated infections in soft tissues and bones remain a
serious complication in orthopedic surgery with infection rates of 1–3% (Harris and Sledge,
1990, Shi et al., 2006, Kurtz et al., 2012, Setyawati et al., 2014). According to the National
Healthcare Safety Network data, the infection rates for the total joint replacements surgeries
are between 1.7–2% in the United Kingdom (UK) (Ridgeway et al., 2005), and 2.3% in the
United States (US) (Edwards et al., 2009), with the highest rate of 15% for ankle replacement
(Gougoulias et al., 2010). Furthermore, infections are 40% more likely from revision surgery
than from the first implantation (Trampuz and Zimmerli, 2005). Conventional treatments
such as systemic antibiotics are expensive ($15,000–$30,000), may be prone to complications
and leading to impaired healing, need for revision surgery and prolonged hospitalization
(Prokopovich et al., 2015, Darouiche, 2004). As a prophylactic measure to reduce the risk of
infections especially bacterial osteomyelitis, the use of drug-loaded implants with local
delivery of antibiotics has become common clinical practice over the last four decades, thus
minimizing the need for follow-up care and improving patient comfort. Antibiotic-loaded
biomaterials and their sustained release over time are expected to yield high antibiotic
concentrations to the local site with reduced toxicity and side-effects, which may not be
achieved by systemic routes (Adams et al., 1992, Gerhart et al., 1993, Frutos Cabanillas et al.,
2000).
Despite the widespread clinical use, there are growing concerns about the clinical
efficacy of incorporating antibiotics into bone cements, whence only limited amounts of
antibiotics (typically ~10%) can be released (Anagnostakos and Kelm, 2009, Shen et al.,
2011, Shen et al., 2016). Moreover, the antibiotic release profile is normally characterized by
a high initial burst release followed by a low, non-therapeutically effective phase (Ensing et
al., 2008), wherein biofilm formation may persist. Since the release mechanisms are poorly
understood and the release rates are generally low, the sub-inhibitory antibiotic
concentrations result over extended periods of time that may culminate in antibiotic
resistance amongst infectious microorganisms (Hope et al., 1989). In addition, the drug-
impregnated bone cements are usually accompanied by a loss in mechanical strength, which
is critical for the weight-bearing cements, rendering them unsuitable for prosthesis fixation in
primary arthroplasty (Klekamp et al., 1999, Lewis and Janna, 2006, Jiranek et al., 2006).
Since the long-term mechanical stability of the acrylic bone cement are crucial factors to
determine its application in orthopedic surgery, the efficacy of existing antibiotic-loaded bone
cements for primary implant fixation may be debatable and should be critically considered.
Mechanical stability is a vital factor that need to be considered since there is a high
possibility for the antibiotic loaded bone cement (with excellent drug delivery profiles) to be
failed clinically due to their poor mechanical properties. More attempts have been undertaken
to formulate bone cements without deleterious effects on the biomechanical properties of the
cements. Puska et al. (2016) investigated the mechanical properties of
polymethylmethacrylate bone cement matrix functionalized with trimethoxysilyl and
bioactive glass. Sheafi and Tanner (2014) tested the effect of shape of bone cements and
surface preparation techniques on the fatigue behaviour of commercial bone cements,
Smartset-GHV and DePuy CMW1. Prokopovich et al. (2013) reported the compressive
strength of cements impregnated with silver–tiopronin nanoparticles. Shen et al. (2011)
conducted three-point bending and compressive tests on MSN-functionalized PMMA bone
cements before antibiotic release. Shi et al. (2006) studied the antibacterial and mechanical
properties of bone cements impregnated with chitosan and ammonium chitosan derivative
nanoparticles. Puska et al. (2003, 2004b) investigated the mechanical properties of oligomer-
modified acrylic bone cement with glass-fibers (Puska et al., 2004a). However, to our
knowledge very limited studies have been reported the biomechanical stability of bone
cements especially after aging for extended periods. Therefore, a comprehensive
understanding of the resulting impact on the cement’s properties is required in order to extend
the use of functionalized and antibiotic-loaded bone cements for load-bearing applications,
such as primary arthroplasty or articulating cement spacers used in revision procedures.
This research aimed to examine the effects of modification of bone cements (Simplex-
P and Smartset-HV) using MSNs and loaded with GTMC on their mechanical properties as
compared with commercially available antibiotic-loaded bone cements (Smartset-GHV).
Properties such as mechanical strength before and after aging for 6 months and antibiotic-
release characteristics have been investigated. It has been reported that water soluble xylitol
and high drug loading could enhance drug elution rate (Slane et al., 2014c, Pithankuakul et
al., 2015), thus bone cements with xylitol and, high drug loadings were formulated and their
mechanical properties were also investigated as a comparison. It was found that the
incorporation of the antibiotics into MSN-functionalized bone cements has minimal effects
on the physical properties even after the elution of the antibiotics.
2. Materials and Methods
2.1. Materials
Gentamicin (GTMC), poly(ethylene glycol)-block-poly(propylene glycol)-block-
poly(ethylene glycol) (Pluronic P123) and tetraethyl orthosilicate (TEOS, 98%) were
purchased from Sigma-Aldrich. Fluorocarbon surfactant FC-4 was purchased from Yick-Vic
Chemicals & Pharmaceuticals (HK) Ltd. Commercial bone cements CMW Smartset-GHV
and Smartset-HV (DePuy International Ltd. UK) were obtained from Johnson & Johnson Pte
Ltd. Simplex-P Radiopaque (Stryker Co, UK) from Stryker Singapore Pte Ltd. All other
reagents and solvents used in the study were of reagent grade and were used without further
purification.
2.2. Synthesis of mesoporous silica nanoparticles
Mesoporous silica nanoparticles (MSNs) were prepared using fluorocarbon-surfactant-
mediated synthesis (Han and Ying, 2004). A total of 0.5 g of Pluronic P123 and 1.4 g of FC-4
were dissolved in 80 ml of 0.02 M HCl solution at 30°C, followed by the introduction of 2.0
g of TEOS under stirring. The solution was continuously stirred at 30°C for 24 h and then
transferred into a polypropylene bottle and kept at 100°C for 24 h. The resultant solid was
recovered by centrifuging and washed with deionized water twice, then dried at 55°C for 12
h. The material was heated from room temperature to 550°C at a heating rate of 2°C/min and
followed by calcination in air for 6 h to remove the template molecules.
2.3. Preparation of antibiotic-loaded bone cements
GTMC-MSNs were loaded by direct impregnation with PMMA-based bone cement powder.
A total of 0.24 g of MSNs was dispersed by ultrasonication in 4 ml of aqueous solution
containing 0.08g of GTMC and aged for 3 h. Subsequently, 1.68 g of Simplex-P bone cement
powder was immersed into the aqueous suspension to form slurry under stirring. The wet
mixture was dried under vacuum at room temperature for more than 1 day. Finally, the dried
GTMC-MSN loaded bone cement was ground to fine powder. In comparison, different types
of drug loaded bone cements were synthesized, of which the compositions are shown in
Table 1. In addition, commercially available antibiotic-loaded bone cements, Smartset-GHV,
was used as a control. The formulated bone cement powders listed in Table 1 were mixed
with liquid monomer in a ratio of 2 g/ml in a laminar flow hood according to the
manufacturer’s instruction. The monomer liquid was added to the PMMA–GTMC-filler
mixture in a bowl and was stirred using a spatula until the powder was fully wetted. The soft
dough-like mixture was inserted into the molds manually. Different molds were used to make
samples for the different tests. Rectangular beams (25 × 10 × 2 mm) were used for the
bending tests, while the cylindrical specimens (6 mm in diameter and 12 mm in height) were
prepared for the antibiotics elution assays and compression test, respectively. The filled mold
was pressed between two glass plates for hardening overnight at room temperature. The
hardened bone cements were removed from the mold and stored at room temperature.
2.4. Characterization
The external and fractured surfaces of the bone cements were examined by a high-resolution
scanning electron microscope (SEM, JSM-6700F, JEOL, Tokyo, Japan) operating at 5 keV
under the secondary electron image (SEI) and lower secondary electron image (LEI) mode.
Prior to analysis, samples were mounted on double-sided adhesive carbon tapes and coated
with gold for 1 min by a sputter coater (Cressington Sputter Coater 208HR, UK). The internal
porous structures of MSNs were observed by high-resolution transmission electron
microscopy (TEM) TECNAI F20 (G2) (FEI, Philips Electron Optics, Holland) electron
microscope at 200 kV. Nitrogen adsorption-desorption isotherms were measured by using an
Autosorb-6B gas adsorption analyzer (Quantachrome Instruments, Boynton Beach, FL) at –
196 °C (77 K). MSNs were degassed under vacuum at 200°C while drug-loaded MSN
samples were outgassed at 40°C for 24 h prior to analysis to remove any residuals and
absorbed water. The specific surface areas of the samples were assessed from the linear
region of the Brumauer-Emmett-Teller (BET) plots. The total pore volume was estimated
from the amount of N2 adsorbed at a relative pressure of 0.95, while mesopore size
distributions were computed from the adsorption branch of N2 adsorption-desorption
isotherms using the conventional Barrett-Joyner-Halenda (BJH) approach. The contact angle
measurement was performed with the sessile-drop technique with contact-angle analyser
KSV CAM 100 (Finland). Approximately 50 mg of bone cement powder samples were
compressed into tablets by a hydraulic press at a pressure of 75 MPa for 1 min. A water
droplet was placed on the compact surface using a microsyringe and photographed to
determine the contact angles. Meanwhile, the silicon composition leached out from bone
cements during aging was quantified by using inductively-coupled plasma optical emission
spectrometry (Varian Vista–MPX CCD Simultaneous ICP-OES). A calibration standard of
silicon was prepared from Inorganic Ventures (USA) calibration stocks (approximately 999 ±
5 µg/mL). Before ICP-OES analysis, the bone cement samples were soaked in 5 ml of PBS
buffer (pH 7.2) in an incubator shaker at 37°C and 40 rpm up to 6 months. All the
measurements were performed at room temperature and three separately prepared samples
were analyzed per cement to ensure reproducibility.
2.5. Antibiotic elution kinetics
The drug-release study was conducted by soaking three cylindrical samples (6 mm in
diameter and 12 mm in height) in 5 ml of PBS buffer (pH 7.2). The sample was kept in an
incubator shaker operated at 37°C and 40 rpm. The release medium was withdrawn at pre-
determined time intervals, and replaced with fresh PBS buffer (5 ml) each time. The
accumulative amount of GTMC released was calculated based on the initial weight of the
bone cement cylinder and the drug content. The GTMC release study was conducted
approximately for 80 days. An indirect method was used for measurement of the GTMC
concentration by a UV–Vis spectrophotometer (Cary 50, Varian Co) because GTMC absorb
neither ultraviolet nor visible lights. The o-phthaldialdehyde was used as a derivatizing agent
to react with the amino groups of GTMC and yield chromophoric products (Zhang et al.,
1994). A total of 1 ml of GTMC solution was reacted with 1 ml of isopropanol (to avoid the
precipitation of the products formed) and 1 ml of o-phthaldialdehyde reagent solution. After
full mixing, the concentration of GTMC was determined by the UV absorbance at 332 nm.
2.6. Testing of mechanical properties
Three-point bending tests were performed on the Zwick/Roell material-testing machine
(Model 5544). According to the standard test method of ASTM D790-3, the span length was
20 mm and the loading rate was 1 mm/min. The bending modulus (EB) was calculated
according to the following equation: EB = L3 m/4bd
3 , where L is the support span (mm), b is
the width of beam tested (mm), d is the depth of beam tested (mm), and m is slope of the
tangent to the initial straight-line portion of the load–deflection curve (N/mm). The
compression tests were carried out on the bone cement cylinders with the same dimensions as
that for the drug-release investigation. The compression force was applied along the axis
using a crosshead speed of 10 mm/min. The compression strength (CS) was calculated from
the obtained load-deformation curves with the following equations: CS = F/A, where F is the
applied load (N) at the highest point of the load–deflection curve and A is the cross-section
area of the sample tested.
2.7. In vitro antibacterial assay
Six experimental groups including plain cements without GTMC (negative control) and
MSN-functionalized antibiotic loaded cements and commercial bone cement (positive
control) were subjected to in vitro antimicrobial assays. All the bone cement pellets with
dimensions of 6 mm in diameter and 12 mm in height were Ethylene Oxide sterilized before
the antibacterial assay. The bacterial species (S. aureus from NCTC 7447) were grown in
Trypton Soya Broth (TSB) media. The sterilized bone cement pellets were incubated with
sterile PBS (5mL each) at 37ºC for 4 weeks with mild shaking. They were then separated
from the eluted PBS and bone cement pellets were individually cultured with 1mL of S.
aureus (1x107 cfu/ml) for three days in TSB media at 37
ºC with mild shaking. Following the
three day period the resultant cultured medium was optically measured using a
spectrophotometer with a plastic cuvette at 600 nm. All experiments were performed in
triplicate.
2.8. Statistical analysis
Data were processed using Microsoft Excel 2003 software. Each sample was tested in
triplicates and the mean ± standard deviation is reported. Two sample comparisons of means
were carried out using Student’s t-test analysis and statistical significance was ascertained
when the p-value was less than 0.05.
3. Results and Discussion
3.1. Characterization
Figure 1 illustrates the morphology of MSNs as revealed by TEM and SEM. The
MSNs appeared in a rod-like morphology with an average size of 100–600 nm in length and
100 nm in diameter. The TEM image shows that the well-dispersed MSNs have highly-
ordered and uniformly-arranged pore channels along the axial direction of the rod-like
nanoparticles. Figure 2(A) displays the N2 adsorption–desorption isotherms and Figure 2(B)
illustrates the pore size distribution of MSNs before and after drug loading. The pure MSNs
show a type IV isotherm and H1 hysteresis and with a high capacity of N2 adsorption, a large
pore volume of 1.33 ± 0.27 cm3/g and a high BET surface area of 761.0 ± 77.3 m
2/g. MSNs
have a uniform pore size distribution with an average pore diameter of approximately 6.2 nm.
This pore diameter of MSNs is wide enough to accommodate GTMC molecules which are
much smaller in size (Figure 3) compared with MSNs pore channels. In addition, the large
surface area and pore volume of MSNs makes it suitable for hosting and for further release of
GTMC molecules. The significant reduction in adsorbed nitrogen, total pore volume and
surface area of MSNs after loading with GTMC (Table S1), suggested that the pore channels
of MSNs were occupied by the antibiotic molecules. As the original PMMA-based bone
cement powder is a non-porous material, most of the GTMC would be entrapped into the
mesoporous structures of MSNs after the impregnation instead of being embedded in the
bone cement matrix. In comparison, Slane et al. (2014a, 2014b) investigated the use of
commercial MSNs as a reinforcement material within Palacos R+G bone cements. The
reported commercial MSNs have lower total pore volume and surface area as compared with
MSNs synthesized in this present study, which might not be suitable for drug loading.
Moreover, the morphology, internal structures, and drug delivery efficiency of the
commercial MSNs was not shown. MSNs without proper pore structure and particle shape
might not build up effective diffusion networks to facilitate drug release. In addition, both the
original bone cements (Simplex-P and Smartset-HV) are fairly hydrophobic with contact
angle around 90⁰ (Table 2). Meanwhile, MSNs are highly hydrophilic, for which no contact
angle can be measured. The addition of MSNs significantly reduced the contact angles as
compared with BC-3, BC-4 and Smartset-GHV, indicating the improvement of wettability of
the MSN-functionalized bone cements.
3.2. In vitro drug-release study
Figure 4 displays the cumulative release profile of GTMC from different types of
formulated bone cements. Bone cements with xylitol (BC-3), high drug loading without
MSNs (BC-4) and Smartset-GHV were used as controls for MSN-functionalized bone
cements. A remarkable enhancement can be observed in the cumulative release of GTMC
from MSN-functionalized bones cement composites. The elution profile of GTMC from
MSN-functionalized bone cement is in a biphasic profile consisting of an initial burst release
followed by a gradual and sustained elution. Therefore, after more than 10% of release in the
first day, a sustained release of GTMC from MSN–bone cements (BC-1 and BC-2) composite
was observed and reached more than 55% of release over the period of 77 days. BC-1
released 73.1% (p < 0.05) and BC-2 was 55.2% (p < 0.05) of loaded GTMC in 77 days. The
prominent GTMC burst effect is advantageous since a high initial antibiotic concentration can
minimize the risk of infection in the immediate post-operative period and sustained release
can further improve the effectiveness of the bone cements for extended period (Frutos et al.,
2010). As reported by Shen et al. (2011, 2016), the magnitude of enhanced and sustained
phase of MSN-functionalized bone cements is highly dependent upon the amount of MSNs
incorporated into the cement matrix which is able to form effective nano-sized diffusion
network pathways for fluid to penetrate and dissolve the GTMC deep within the cement. The
similar amount of MSNs reported by Shen et al. (2011), which is 8.15 wt%, was used in this
present study. In addition, the well-controlled and sustained release of GTMC from MSN-
functionalized bone cements was contributed by the nano-sized pore channels of MSNs
which act as a limiting factor for the diffusion of the GTMC. Slane et al. (2014b) reported the
performance of bone cements, including static mechanical and fatigue test, with maximum
loading of commercially available MSNs of 5 wt%. However, according to our previous
report (Shen et al., 2011), less than 6 wt% of MSNs did not exhibit an obvious improvement
in the release of antibiotics. Most of the loaded MSNs at the low concentration were isolated
and embedded in the bone cement matrix without build effective nano-diffusion networks.
Therefore, a critical content of MSNs is required to build up the network inside the bone
cement for antibiotics to diffuse from matrix to medium.
On the other hand, the commercially available antibiotic-loaded bone cement
(Smartset-GHV) exhibits the lowest GTMC release rate compared with other bone cements.
Smartset-GHV shows GTMC release of only about 6.1% throughout 77 days of immersion in
PBS and no significant release was detected after the first day. Similarly, the bone cement
powder prepared with xylitol (BC-3) and high loading of GTMC without fillers (BC-4) also
did not show significant enhancement in drug release, although higher drug release profile
than Smartset-GHV was observed. Only about 10.5% and 16.8% of GTMC released have
been observed for BC-3 and BC-4, respectively, for 77 days. Slane et al. (2014c) have
reported enhancement of antibiotic elution from Palacos R+G bone cement using different
xylitol loadings, whereby high content of xylitol (14.4 wt%) was required in order to achieve
maximum cumulative release of 41.3% of GTMC in 45 days.
The kinetics of antibiotic release in these cements are controlled by the surface
phenomenon and the total amount released depends on wettability and bulk porosity
(Chapman and Hadley, 1976, Lewis and Janna, 2004). The hydrophobic nature and poor
wettability of the bone cements (Table 3) limited the diffusion of the aqueous dissolution
medium inside the hydrophobic matrix caused only less than 17% of the incorporated
antibiotics to be released. Only surface-adhered GTMC particles could be released and those
GTMC particles embedded in the superficial layers of the PMMA matrix during
polymerization could not diffuse to the surface of the bone cement to be release into the
medium.
3.3. Compression and three-point bending
Figure 5 display the mechanical properties of formulated bone cements before and
after aging for 6 months. Both the original bone cements (Simplex-P and Smartset-HV) have
compression strength of more than 89 MPa and bending modulus of more than 6.7 GPa. The
incorporation of MSNs did not have a significant detrimental impact on the biomechanical
strength as compared with the original bone cements (p < 0.05). The BC-1 and BC-2 preserve
at least 80% of the original bone cement strength, for both of which the compressive strength
is more than 75 MPa after 6 months of aging and the bending modulus is above 5GPa.
Despite the slight compromise by the incorporation of MSNs, the bending modulus and the
compressive strength of MSN-functionalized bone cements are almost similar to the
commercially available antibiotic-loaded bone cement (Smartset-GHV) throughout of 6
months of aging. Slane et al. (2014b) reported the static and fatigue properties of acrylic bone
cement functionalized with various loadings of commercial MSNs (0.5, 2 and 5 wt%) and
found a general decrease in several mechanical properties with increasing MSNs content.
Importantly, all the tests in their report have been conducted on the freshly prepared samples
and no aged samples were investigated. In this present study, good and acceptable
mechanical properties can be observed for acrylic bone cement modified with 8.15 wt% of
MSNs even after been aged for 6 months. In the meantime, BC-3 and BC-4 have shown a
reduction of more than 20% in both the compressive strength and bending modulus within the
first month of aging (data not shown) relative to original Simplex-P. Importantly, the
compressive strength of the bone cements fell below 70 MPa (which is required standard in
ASTM F541 and ISO 5833) after one month of aging.
In addition, mechanical testes on MSN-functionalized bone cements with high-dose
antibiotic loading (BC-5) and binary systems (BC-6 and BC-7) have been investigated.
Recently, both binary and high-dose antibiotic-loaded bone cement were shown great interest
in clinical application to cope with antibiotic-resisted microbes. Since numerous studies have
been reported about the drug release of binary systems previously (Penner et al., 1996,
Cerretani et al., 2002, Duey et al., 2012), in this study more attention has been given to their
mechanical properties. The compositions of the bone cements have been summarized in
Table 3. It is likewise observed that the incorporation of MSNs incurs negligible effects on
the mechanical properties of these bone cements (Figure 6). Although some decrease in
mechanical properties can be observed as compared with the single-drug systems, all the
formulated samples nonetheless demonstrate mechanical strength greater than that of the
ASTM F541 and ISO 5833 standard.
The functionalization of bone cements with MSNs incurs negligible effects on the
mechanical properties. No significant changes in the PMMA-based bone cement structures
[which appeared to be in condensed and nonporous forms (Figure S1)] could be observed due
to the incorporation of MSNs. Since most of the antibiotic molecules were entrapped in the
pore channels and released from the pore channels of MSNs, no addition voids have been
created in the PMMA cement matrix. In addition, the ICP-OES results show that less than
3.52% of MSNs was leached out from bone cements throughout the 6 months of aging. The
MSNs remain in the matrix and supports the structures of the bone cements, thus maintaining
their mechanical properties after the drug release. Puska et al. have reported the importance
of bone-bonding between the filler particles and matrix in order to preserve good mechanical
properties (Puska et al., 2016). Meanwhile, the good mechanical strength of commercially
available Smartset-GHV might be due to the unchanged structure of the Smartset-GHV
(Figure S1) as a result of the poor GTMC release. More than 90% of GTMC remained
entrapped in the bone cement matrix which able to maintain the cement's structure. However,
significant changes in the cement structures (with a large number of micron-sized voids)
could be observed after the aging for BC-3 and BC-4 (Figure 7). As reported previously, the
poor mechanical strength of BC-3 and BC-4 are mainly due to the presence of large portions
of micron-sized voids and cracks in the bone cement created by the release of antibiotics and
xylitol (van de Belt et al., 2000, Slane et al., 2014c). In contrast, Puska et al. (2007) reported
that the porosity of bone cements have potential in creating enhanced biological fixation
between the cement and the bone tissue, whereby the porous structures may adhere
biologically to the surrounding bones and allow the bone ingrowth into the cement layer.
3.4. In vitro antibacterial assay
The sustainable antibacterial property of MSN-functionalized antibiotics bone
cements was compared with plain bone cements and commercial Smartset-GHV (Figure S2).
After drug release in PBS solution for 4 weeks, the viability of S. aureus is still obviously
being inhibited by BC-1 and BC-2 as compared with Smartset-GHV. The antibacterial
property of BC-1 and BC-2 still can be preserved up to 4 weeks, even though their
antibacterial effectiveness is decreased as compared with freshly prepared samples due to the
loss of gentamicin into the PBS buffer solution. This sustainable antibiotic property of MSN-
functionalized bone cements is attributed the continuous release of GTMC for extended
periods, thus could largely prevent relapse or recurrence of bacterial infection after
orthopaedic surgery. As comparison, the commercial Smartset-GHV bone cement almost
completely loses its antibacterial efficacy after 4 weeks immersion in PBS and shows similar
bacterial viability as plain bone cements. This is because the Smartset-GHV has negligible
antibiotic release after the first day of immersion in PBS buffer (Figure 4). The antibiotics
embedded in the bone cement cannot diffuse to the external surface of the bone cement, and
thus almost all bacterial could be viable.
4. Conclusions
MSNs incorporation in bone cement enabled efficient and sustained delivery of antibiotics.
The loaded MSNs afforded an effective nano-sized diffusion network in the acrylic bone
cement matrix, which was responsible for the effective drug diffusion and extended time-
release to the external surfaces. The effect of MSNs on mechanical properties was
investigated before and after aging in PBS buffer solution for up to 6 months. The results
indicate that the PMMA-based bone cements functionalized with MSNs demonstrated
improved antibiotic release without inducing deleterious effects on the weight-bearing
mechanical properties of the cements. Negligible negative effect on the mechanical properties
of the bone cements was detected, even at the high drug loading and binary antibiotic systems
tested. ICP measurement indicated that more than 96% of MSNs was remained in bone
cement after aging in PBS for 6 months. The presence of MSNs in bone cement matrix after
the drug release or aging is believed to support the bone cements structure, thus preserve their
mechanical strength. The MSN-functionalized bone cements exhibited sustainable
antibacterial activity against S. aureus after immersion in PBS solution for 4 weeks. As
comparison, soluble xylitol-modified and high-dose antibiotic-loaded bone cements showed
limited enhancement in antibiotic release and substantially negative influence on the
mechanical properties of the bone cements. Furthermore, the mechanical strength was
seriously impaired after the drug release as more voids could be formed due to leaching of
soluble polymer or release of antibiotic.
6. Disclosure
The authors report no conflicts of interest in this work.
Acknowledgement
This work was generously supported by Biomedical Engineering Programme (BEP) 2014
Grant (ICES/14-422A02), the Institute of Chemical Engineering and Sciences, and the
Agency of Science Technology and Research (A*STAR), Singapore.
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List of Figure Captions
Figure 1. Morphology of MSNs: (i) SEM and (ii) TEM image
Figure 2. (A) N2 adsorption-desorption isotherms and (B) pore size distribution of MSNs
before and after encapsulation with GTMC
Figure 3. 3D molecular structure and molecular size of GTMC (Doadrio et al., 2004)
Figure 4. Cumulative GTMC-release profile from modified PMMA-based bone cements
formulated with MSNs (BC-1, BC-2), with xylitol (BC-3), and with higher drug loading
without MSNs (BC-4), and commercial antibiotic-loaded Smartset GHV bone cement as a
comparison
Figure 5. (A) Compression strength and (B) bending modulus of PMMA-based bone
cements before and after aging for different periods
Figure 6. (A) Compression strength and (B) bending modulus of PMMA-based bone cement
with high drug loading of GTMC and combination of two antibiotics.
Figure 7. SEM images of (A) BC-1, (B) BC-3 and (C) BC-4: (i) before and (ii) after GTMC
released for 2 months. Note: Bar is equal to 1 µm.
Tables
Table 1 Composition of nanomaterial-formulated antibiotic bone cements.
Denotation Bone cement PMMA bone cement GTMC
(g)
MMA
(ml)
Fillers (g) Drug
Loading
(%)
Simplex-
P (g)
Smartset-
HV (g)
MSN Xylitol
BC-1 GTMC/MSN/Simplex-P
(2.72 wt%)
1.68 - 0.08 1.0 0.24 - 2.72
BC-2 GTMC/MSN/Smartset-HV
(2.72 wt%)
- 1.68 0.08 1.0 0.24 - 2.72
BC-3 GTMC/Xylitol/Simplex-P
(2.72 wt%)
1.68 - 0.08 1.0 - 0.24 2.72
BC-4 GTMC/Simplex-P
(10 wt%)
1.70 0.30 1.0 - - 10
Table 2. Water contact angles of the deferent functionalized-loaded bone cements employed
in this study. The values are expressed as mean ± SD
Bone cement Contact angle (degrees)
MSN Not measurable
Simplex-P 88.6 ± 5.8
Smartset-HV 99.5 ± 4.4
BC-1 26.6 ± 2.3
BC-2 19.7 ± 2.7
BC-3 50.3 ± 1.4
BC-4 69.8 ± 0.7
Smartset-GHV 88.2 ± 6.5
Table 3 Composition of nanomaterial-formulated antibiotic bone cements.
Denotation Bone cement MSN Antibiotics (g) MMA
(ml)
MSN Drug
Loading
(%) GTMC VCMC TBMC
BC-5 GTMC/MSN/Simplex-P
(8.15 wt%)
0.24 0.24 - - 1.0 0.24 8.15
BC-6 GTMC/VCMC/MSN/Simplex-P
(5.44 wt%)
0.24 0.08 0.08 - 1.0 0.24 5.44
BC-7 GTMC/TBMC/MSN/Simplex-P
(5.44 wt%)
0.24 0.08 - 0.08 1.0 0.24 5.44
Note: VCMC stands for vancomycin; TBMC stands for tobramycin.
Highlights
1. MSN-functionalized bone cements enable efficient and sustained delivery of GTMC.
2. MSNs afforded an effective nano-sized diffusion network in the bone cements.
3. Loaded MSNs shows no effects on the biomechanical properties of the bone cements.
4. More than 96% of MSNs remains in the matrix to support the cement structures.
5. MSN-functionalized cements exhibited sustainable antibacterial activity.
List of Figure Captions
Figure 1. Morphology of MSNs: (i) SEM and (ii) TEM image
Figure 2. (A) N2 adsorption-desorption isotherms and (B) pore size distribution of MSNs
before and after encapsulation with GTMC
Figure 3. 3D molecular structure and molecular size of GTMC (Doadrio et al., 2004)
Figure 4. Cumulative GTMC-release profile from modified PMMA-based bone cements
formulated with MSNs (BC-1, BC-2), with xylitol (BC-3), and with higher drug loading
without MSNs (BC-4), and commercial antibiotic-loaded Smartset GHV bone cement as a
comparison
Figure 5. (A) Compression strength and (B) bending modulus of PMMA-based bone
cements before and after aging for different periods
Figure 6. (A) Compression strength and (B) bending modulus of PMMA-based bone cement
with high drug loading of GTMC and combination of two antibiotics.
Figure 7. SEM images of (A) BC-1, (B) BC-3 and (C) BC-4: (i) before and (ii) after GTMC
released for 2 months. Note: Bar is equal to 1 µm.
Figure 1. Morphology of MSNs: (i) SEM and (ii) TEM image
Figure 2. (A) N2 adsorption-desorption isotherms and (B) pore size distribution of MSNs
before and after encapsulation with GTMC
Figure 3. 3D molecular structure and molecular size of GTMC (Doadrio et al., 2004)
Figure 4. Cumulative GTMC-release profile from modified PMMA-based bone cements
formulated with MSNs (BC-1, BC-2), with xylitol (BC-3), and with higher drug loading
without MSNs (BC-4), and commercial antibiotic-loaded Smartset GHV bone cement as a
comparison
Figure 5. (A) Compression strength and (B) bending modulus of PMMA-based bone
cements before and after aging for different periods
Figure 6. (A) Compression strength and (B) bending modulus of PMMA-based bone cement
with high drug loading of GTMC and combination of two antibiotics.
Figure 7. SEM images of (A) BC-1, (B) BC-3 and (C) BC-4: (i) before and (ii) after GTMC
released for 2 months. Note: Bar is equal to 1 µm.
Graphical Abstract
