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RESEARCH ARTICLE
Experimental model of peri-prosthetic infection of the knee caused
by Staphylococcus aureus using biomaterials representative
of modern TKA
Jodie L. Morris
1,2,
*, Hayley L. Letson
2
, Andrea Grant
1
, Matthew Wilkinson
1
, Kaushik Hazratwala
1
and Peter McEwen
1
ABSTRACT
Prosthetic joint infection (PJI) following total knee arthroplasty (TKA)
remains the leading cause for revision surgery, with Staphylococcus
aureus the bacterium most frequently responsible. We describe a
novel rat model of implant-associated S. aureus infection of the knee
using orthopaedic materials relevant to modern TKA. Male Sprague-
Dawley rats underwent unilateral knee implant surgery, which
involved placement of a cementless, porous titanium implant into
the femur, and an ultra-highly cross-linked polyethyelene (UHXLPE)
implant into the proximal tibia within a mantle of gentamicin-laden
bone cement. S. aureus biofilms were established on the surface of
titanium implants prior to implantation into the femur of infected
animals, whilst control animals received sterile implants. Compared
to controls, the time taken to full weight-bear and recover pre-
surgical body weight was greater in the infected group. Neutrophils
and C-reactive protein levels were significantly higher in infected
compared to control animals at day 5 post surgery, returning
to baseline levels for the remainder of the 28-day experimental
period. Blood cultures remained negative and additional plasma
inflammatory markers were comparable for control and infected
animals, consistent with the clinical presentation of delayed-onset
PJI. S. aureus was recovered from joint tissue and implants at day 28
post surgery from all animals that received pre-seeded titanium
implants, despite the use of antibiotic-laden cement. Persistent
localised infection was associated with increased inflammatory
responses and radiological changes in peri-implant tissue. The
availability of a preclinical model that is reproducible based on the
use of current TKA materials and consistent with clinical features of
delayed-onset PJI will be valuable for evaluation of innovative
therapeutic approaches.
KEY WORDS: Animal model, Prosthetic joint infection, Biofilm,
Inflammation, Staphylococcus aureus, Total knee arthroplasty
INTRODUCTION
Currently, more than 4.7 million and 600,000 people in the USA and
Australia, respectively, are estimated to be living with a total knee
arthroplasty (TKA), with conservative projections estimating a 143%
increase in incidence rates of TKA by 2050 (Maradit Kremers
et al., 2015; AOANJRR, 2017; Inacio et al., 2017). Significant
advancements to preoperative and surgical protocols and orthopaedic
materials have reduced postoperative infection rates to less than 2%,
however peri-prosthetic joint infection (PJI) remains the leading
cause of implant failure following TKA (Springer et al., 2017; Tande
and Patel, 2014). Methicillin-sensitive Staphylococcus aureus
(MSSA) is the most common cause of PJI following TKA (Guo
et al., 2017; Tande and Patel, 2014). Diagnosis and treatment of PJI
poses a significant burden on both the patient and healthcare system,
with eradication typically requiring multiple surgical interventions,
prolonged hospitalisation, and aggressive and extensive antibiotic
therapy (Beam and Osmon, 2018; King et al., 2018; Tande et al.,
2017).
PJI are broadly classified according to the time from arthroplasty
to development of infection (Beam and Osmon, 2018). Early-onset
PJIs are defined as those occurring within 3 months of surgery, and
arise due to intraoperative contamination with either a large bacterial
burden or a virulent bacterial strain. Delayed-onset PJIs result from
the introduction of less virulent microbes during surgery, and as
such tend to become clinically apparent between 3 months to 1 year
post surgery. In contrast, late-onset PJIs present more than 1 year
post surgery and are frequently due to haematogenous seeding of the
implanted joint from a distant site of infection. Delayed and late-
onset PJIs typically involve implant-associated biofilms (Beam and
Osmon, 2018).
Bio-inert orthopaedic materials such as titanium provide habitable
substrates for biofilm formation, a growth state which serves to
facilitate bacterial survival in hostile environments (Kostakioti et al.,
2013; Ricciardi et al., 2018). Colonisation of an implant begins with
adhesion of planktonic (free-floating) bacteria to the implant surface,
upregulation of genes that facilitate a sessile lifestyle, proliferation
and aggregation of bacterial cells into micro-colonies. The bacterial
aggregates produce extracellular polymeric substances (EPS), at
which point bacterial attachment becomes irreversible. Subsequent
maturation of the biofilm is regulated by highly sophisticated,
intercellular signalling networks and involves the development of a
multi-layered, three-dimensional (3D) microbial community encased
within a self-produced matrix of carbohydrate-rich polymers,
proteins and nucleic acids (Ricciardi et al., 2018). Detachment and
dispersal of planktonic bacterial cells from the periphery of mature
biofilms facilitates dissemination and seeding of distant sites
(Kostakioti et al., 2013). The metabolic activity of bacterial cells
within a biofilm varies across a spectrum that inversely corresponds
Received 29 May 2019; Accepted 30 August 2019
1
Orthopaedic Research Institute of Queensland, Townsville 4812, Australia.
2
College of Medicine, Division of Tropical Health and Medicine, James Cook
University, Townsville 4811, Australia.
*Author for correspondence (jodie.morris@oriql.com.au)
J.L.M., 0000-0002-4795-5539; H.L.L., 0000-0003-0135-134X; A.G., 0000-0002-
0568-0872; M.W., 0000-0002-3704-7043; K.H., 0000-0002-0649-9231; P.M., 0000-
0001-5499-9532
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
1
© 2019. Published by The Company of Biologists Ltd
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to nutrient availability, with those closest to the implant surface
exhibiting metabolic inactivity and slower growth rates (Kostakioti
et al., 2013). As such, implant-associated biofilms serve to protect
bacteria from the host immune response and antibiotics, facilitating
persistent infection and increased likelihood for the emergence of
antibiotic-resistant bacterial strains (Arciola et al., 2018; Ricciardi
et al., 2018). Often characteristic signs and symptoms of bacterial
infection,such as elevated systemic inflammatory markers,are absent
since bacteria within the biofilm are shielded from host immune
responses (Beam and Osmon, 2018). Further, diagnosis of biofilm-
associated PJI is also problematic due to the difficulty in removal and
culture of bacterial cells from mature biofilms using conventional
microbiological methods (Arciola et al., 2018; Beam and Osmon,
2018).
The difficulty preventing, diagnosing and eradicating biofilm-
associated PJI, and the continued emergence of bacterial resistance
to conventional and current antibiotics, is driving global interest in
development of innovative therapeutic approaches (Li and Webster,
2018; Osmon, 2017). Clinically relevant small animal models
are essential for preclinical evaluation of new preventative and
therapeutic strategies. Several rodent models of implant-related
S. aureus osteomyelitis have been described (Edelstein et al., 2017;
Lucke et al., 2003; Reizner et al., 2014; Schindeler et al., 2018; Søe
et al., 2013). However, many are based on the use of orthopaedic
materials that do not reflect current clinical practice for arthroplasty,
thus potentially limiting the translational capacity of findings. To
address this, we sought to develop an experimental model of
delayed-onset PJI caused by MSSA using current and clinically
relevant TKA biomaterials.
In modern TKA, hybrid fixation techniques involving a
cementless femoral component and a cemented tibial component
is often used (Vertullo et al., 2018). Titanium alloys are one
of the most commonly used metals in non-bearing surfaces
of orthopaedic implants, whilst ultra-highly cross-linked
polyethyelene (UHXLPE) is used for articulating surfaces
(Bravin and Dietz, 2018). To reflect this combination of
biomaterials and surgical techniques, we developed a rat model
of knee implant surgery using a 3D-printed porous titanium
implant that is press-fit into the femur, and a cemented UHXLPE
tibial implant. We then progressed this surgical model to one
representative of delayed-onset PJI caused by MSSA, using a
previously characterised clinical strain from a patient with post-
TKA PJI. The biofilm-forming capacity of the MSSA strain on the
titanium implants used in the current study was recently
demonstrated (Morris et al., 2018). Bacterial surface adhesion
and irreversible attachment is a pivotal step in implant colonisation
and establishment of persistent infection (Arciola et al., 2018). To
ensure consistency in the number of implant-adherent bacteria, and
therefore consistency and reliability of the experimental infection
model, titanium scaffolds were pre-seeded with MSSA prior to
implantation. Akin to the features of delayed-onset PJI, we
demonstrate the establishment of a persistent infection that is
localised to the implanted knee in all animals at 4 weeks after
surgery, in the absence of bacteraemia and systemic inflammation.
RESULTS
Clinical outcomes
Radiographs at day 7 post surgery confirmed the titanium and
polyethylene implants were appropriately seated and stable within
the femoral and tibial canals, respectively (Fig. S1A). All animals
survived surgery and the postoperative period, with no signs of
systemic illness. Based on improved clinical scores, pain relief was
ceased for all animals by day 5 post surgery with no adverse clinical
effects observed through the remainder of the experimental
period. No significant differences were observed between body
temperatures of control and infected animals throughout the
experimental period (Fig. 1A). Control animals were able to
partially bear weight on the operated limb within 48 h, with the
median time to full weight-bearing 4 days post surgery (range,
3–6 days). In contrast, while S. aureus-infected animals were able to
bear partial weight within 48 h of surgery, the median time to full
weight-bear was significantly greater than control animals (4 versus
26 days post surgery), with four of eight animals not returning to full
weight-bearing within the 28-day experimental period (P<0.001).
Despite minor weight loss in the first week following surgery, all
control animals returned to and exceeded pre-surgical weights
within 21 days post surgery (Fig. 1B). In contrast, the time taken for
animals in the infected group to return to pre-surgical body weight
was delayed (control, 16.9±4.6 versus infection, 23.3±
5.0 days post surgery, P=0.019; Fig. 1B).
Fig. 1. Changes in body temperature and weight in control (n=12) and infected (n=13) animals following knee implant surgery. Data show mean±
s.e.m. *P<0.05 compared to control animals, two-way ANOVA with Holm-Sidak multiple comparison test.
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Haematology and systemic inflammation
There was no statistically significant difference in baseline
haematology parameters assessed for control and infected animals
(Table 1). Similarly, no significant differences were observed in
red blood cell parameters between control and infected animals
throughout the experimental period. However, differences were
observed in the white blood cell differential counts for control and
infected animals (Table 1). The percentage of lymphocytes was
significantly lower for infected animals at day 5 post surgery
compared to controls (P=0.004). By day 28 post surgery, the
proportion of lymphocytes was higher than baseline levels for both
the control and infected group (P=0.032 and P=0.02, respectively).
At day 5 post surgery, a significantly higher proportion of
granulocytes was observed in blood from the infected group
compared to controls, with levels returning to baseline range by day
10 post surgery (P<0.001; Table 1). Compared to baseline, total
leucocyte numbers were lower at day 28 post surgery in infected
animals (P=0.006), with a similar trend observed for control animals
although this did not reach significance (P=0.07). Significant
decreases were also observed in the total number and percentage of
circulating monocytes in infected animals at day 10 post surgery,
with levels remaining lower than baseline values at the end of the
experimental period (Table 1).
Erythrocyte sedimentation rate (ESR) was comparable for control
and infected animals at day 28 post surgery (Table 1). Plasma
C-reactive protein (CRP) concentrations in control animals
remained unchanged over the 28-day period. In contrast, plasma
CRP levels were increased in infected animals at day 5 post surgery,
though concentrations returned to baseline levels by day 10 post
surgery (P<0.001; Table 1). No significant differences were
observed in plasma inflammatory chemokine and cytokine levels
between control and infected animals across the experimental period
(Fig. 2). TNF-αand IFN-γlevels remained at the assay limit of
detection throughout the experimental period (data not shown).
Plasma IL-10 levels were significantly higher at day 5 post surgery
compared to baseline in both control and infected animals, returning
to baseline levels by day 28 post surgery (P=0.033 and P=0.008,
respectively; Fig. 2E). While there was a trend for increased IL-6
and IL-12p70 concentrations in plasma of infected animals at day 28
post surgery compared to controls, this did not reach statistical
significance (Fig. 2C,D).
Joint gross pathology
Surgical incisions healed without complication in control and
infected animals (Fig. S1B,C). Upon dissection, macroscopic
examination of the operated knees of control animals at 28 days
post surgery revealed mild soft tissue damage and clear synovial
fluid (Fig. 3A,E), with increased joint circumference compared to
the non-operated left knee (P=0.03; Fig. 3D). In contrast, mild-to-
moderate soft tissue and articular cartilage damage was evident
within joints of infected animals, often in combination with
increased viscosity and amounts of synovial fluid (Fig. 3B,C,F–H).
Joint circumferences of implanted knees were comparable between
control and infected animals (P=0.12; Fig. 3D).
Table 1. Haematology parameters
Indices
Time post surgery (days)
P-value0 5 10 20 28
RBC, ×10
12
cells/l Control 7.7±0.3 7.5±0.3 7.1±0.3 7.6±0.3 7.7±0.1 0.162
Infected 7.9±0.4 7.5±0.5 7.5±0.5 7.5±0.4 7.7±0.2
Hgb, g/dl Control 13.6±0.5 12.9±0.8 12.6±0.8 13.5±0.5 13.6±0.4 0.530
Infected 13.6±0.9 13.0±0.8 13.1±0.9 13.3±0.5 13.3±0.7
Hematocrit, % Control 41.6±2.3 41.3±2.6 38.9±1.9 42.8±1.1 42.6±1.7 0.140
Infected 42.6±1.7 40.7±2.8 40.2±2.6 41.3±1.1 40.9±2.3
MCV, fL Control 54.0±2.3 55.0±2.3 54.6±1.1 56.5±1.3 55.1±1.9 0.443
Infected 54.3±2.1 54.0±1.3 53.4±1.5 54.8±2.3 53.4±2.9
MCH, pg Control 17.6±0.3 17.2±0.6 17.7±0.5 17.8±0.5 17.6±0.3 0.600
Infected 17.3±0.5 17.3±0.5 17.4±0.6 17.6±0.4 17.4±0.7
MCHC, g/dl Control 32.7±1.7 31.3±1.4 32.4±1.1 31.5±0.8 32.0±1.5 0.650
Infected 32.6±1.4 32.0±1.0 32.5±1.4 32.1±0.8 32.6±1.3
WBC, ×10
9
cells/l Control 11.0±4.1 12.1±3.9 11.7±5.2 9.4±2.0 7.4±1.3 0.198
Infected 14.6±4.1 11.3±4.7 10.5±2.7 10.3±2.6 8.4±3.1
#
Lymphocytes, ×10
9
cells/l % Control 7.5±2.0 8.0±2.2 8.3±3.2 6.7±1.3 5.7±1.2
#
0.030*
Infected 9.9±2.7 6.2±2.6 7.8±1.0 7.4±1.7 6.5±2.1
#
Control 71.2±11.8 67.9±6.9 73.2±7.2 72.1±6.1 77.0±3.2 0.008*
Infected 68.1±6.2 54.2±9.8*
,#
76.2±10.1 73.0±8.6 77.9±3.6
#
Monocytes, ×10
9
cells/l % Control 0.5±0.7 0.6±0.5 0.6±0.9 0.4±0.6 0.1±0.1 0.274
Infected 1.1±0.7 0.8±0.7 0.3±0.5
#
0.4±0.4 0.2±0.3
Control 3.6±4.5 4.9±3.8 3.8±5.1 3.6±5.6 1.9±1.6 0.357
Infected 7.6±3.7 6.3±3.7 2.2±3.4
#
3.6±4.5 2.1±2.1
Granulocytes, ×10
9
cells/l % Control 3.0±2.0 3.4±1.7 2.8±1.4 2.3±0.7 1.6±0.3 0.542
Infected 3.7±1.1 4.4±1.8 2.4±1.4 2.5±1.1 1.7±0.8
#
Control 25.2±8.0 27.2±6.4 23.0±3.1 24.2±4.9 21.1±2.3 <0.001*
Infected 24.3±3.6 39.5±7.5*
,#
21.6±7.4 23.5±5.3 20.0±2.2
ESR, mm/h Control - - - - 0.69±0.2 0.371
Infected - - - - 0.80±0.3
CRP, µg/ml Control 392.9±165.3 456.1±140.0 436.8±116.1 549.4±159.3 715.6±198.2 0.011*
Infected 417.2±52.3 804.5±223.1*
,#
455.2±159.3 517.21±68.5 585.5±149.8
Values represent mean±s.d. RBC, red blood cell count; Hgb, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC,
mean corpuscular haemoglobin concentration; WBC, white blood cell count; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. Significant difference
between control and infected group, *P<0.05, two-way ANOVA with Holm-Sidak multiple comparison test. Within-subject significant differences from baseline,
#
P<0.05, repeated-measures one-way ANOVA with Dunnett’s multiple comparison test.
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Evaluation of implant stability
Bone ingrowth was evident macroscopically within titanium
implants excised from control animals at day 14 post surgery
(Fig. S2). Micro-computed tomography (MicroCT) analysis of
uninfected, control knees at day 7, 14 and 28 post surgery confirmed
that both the titanium and UHXLPE implants were stable and well-
positioned (Fig. S2). Increases in bone volume (BV) surrounding the
press-fit titanium implant was evident between day 7 and 14 post
surgery (P=0.041) in control animals, followed by a decrease from
day 14 to 28 post surgery (P<0.001; Figs S2 and S3). Similarly, bone-
implant contact (BIC) for the titanium implant increased in the first 2
weeks (P=0.002), then decreased slightly by day 28 post surgery,
though BIC remained significantly higher than 7 days after surgery
(63.7% versus 72%, P=0.02; Figs S2 and S3). Bone parameters were
not quantitatively assessed for the tibial UHXLPE implant since the
bone cement mantle it was constrained within typically filled >80%
of the tibial metaphyseal area. However, new bone formation and
bone-cement contact was evident from histological and microCT
images, with no tibial implant loosening observed in either the
control or infected animals at day 28 post surgery (Fig. S2).
Compared to control animals, BV and BIC were significantly lower
surrounding the femoral titanium implant in infected animals at day
28 post surgery, with evidence of peri-implant osteolysis and bone
remodelling in all animals assessed (Fig. 4).
Fig. 2. Inflammatory chemokine and cytokines in plasma of control (n=5) and infected (n=10) animals at baseline, day 5, 20 and 28 post surgery.
Data show mean±s.e.m. *P<0.05 compared to baseline,
#
P<0.05 compared to day 5 post surgery. Between-group comparisons, two-way ANOVA with Holm-
Sidak multiple comparison test. Within-group comparisons, repeated-measures one-way ANOVA with Dunnett’s multiple comparison test.
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Microbiology
Sterility of the surgical site in control animals was confirmed by the
absence of bacteria in cultures from tissue homogenates of the
spleen, draining lymph nodes, femur, tibia, patella and soft tissue of
the operated knee at day 28 post surgery (Table 2). Similarly, no
bacteria were recovered from the titanium or UHXLPE implants
removed from control animals.
Blood cultures from infected animals remained negative
throughout the experimental period and at end-point analysis.
Similarly, no bacteria were recovered from spleen or liver of
animals in the infected group, with tissue weights comparable to
control animals (Table 2). Bone and joint tissue weights of dissected
knees were also comparable between control and infected animals. At
day 28 post surgery, S. aureus was cultured from joint tissue and
Fig. 3. Joint pathology and bacterial burden. (A–C) Representative images of the operated hind limb of (A) control and (B,C) infected animals at day 28
post surgery.(D) Joint circumference of the non-operated (no implant) and operated (implant) limb of control (n=8) and infected animals (n=13) at day 28
post surgery. Data show mean±s.e.m. (not significant, P=0.12, Student’st-test; *P<0.05 compared to non-operated limb). (E–H) Representative images of
dissected knee of (E) control and (F–H) infected animals at day 28 post surgery. (I) S. aureus was recovered from joint bone and soft tissue, and titanium
implants of all animals in the infected group at day 28 post surgery. Data show mean±s.e.m.
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implants of all animals that had received pre-seeded titanium
implants. Bacterial loads were highest for titanium implants (range,
2.5×10
2
–3.2×10
5
CFU) and femur (range, 55–8.5×10
3
CFU).
Bacteria were recovered from tibia, patella and surrounding
capsular tissue in five of eight infected animals, with considerable
variability in bacterial numbers in these tissues between animals
(Table 2). S. aureus was also cultured from draining lymph nodes of
the implanted knees of three of eight animals at day 28 post surgery
(mean, 98 CFU).
Joint inflammation and histopathology
Compared to control animals, IL-1βwas significantly higher in joint
tissue of infected animals at sacrifice (P=0.013; Fig. 5D). While
differences were not statistically significant, there was a trend for
increased levels of calprotectin, MCP-1 and IL-6 in joints of
infected compared to control animals (Fig. 5A,B,E). In contrast,
levels of TNF-α, IFN-γ, IL-10 and IL-4 tended to be lower in joint
tissue from infected compared to control animals, though these
differences were not statistically significant (ns, Fig. 5).
Joint histopathology was consistent with gross pathology,
inflammatory cytokine and microCT findings. Direct bone contact
with the titanium implant was evident in the femur of control
animals, with new, non-mineralised bone formation at the proximal
and distal zones of the implant (Fig. 6A). There was no evidence of
peri-implant inflammation or osteolysis in sections from control
animals at day 28 post surgery (Fig. 6E,G). Similarly, no
inflammatory changes were observed in synovial tissue from
knees of control animals (Fig. 6C). In contrast, synovial hyperplasia
and infiltration of inflammatory cells into joint capsule tissue was
observed in infected animals (Fig. 6D). Small foci of gram-positive
cocci were also occasionally observed within joint synovial tissue of
infected animals (Fig. 6D, inset). Destruction of normal bone
architecture, woven bone formation and fibrosis was evident in peri-
implant tissue of the femur, with increased inflammatory cell
infiltration and small focal areas of lysis in animals infected with
S. aureus (Fig. 6B,F,H). Compared to control animals, no obvious
histopathological differences were observed within the tibia of
infected animals.
Fig. 4. Implant stability. (A–D) Representative axial (A,B)
and coronal (C,D) microCT scans of the distal femur in
control and infected animals at day 28 post surgery.
(E) The bone volume (BV) percentage within a 55,
150 and 300 µm distance from the implant surface, and
bone-implant contact (BIC) percentage for the press-fit
titanium implant was significantly lower in infected (n=5)
compared to control animals (n=5) at day 28 post surgery.
Data show median±i.q.r. **P<0.01, Mann–Whitney test.
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DISCUSSION
Implant failure due to bacterial infection continues to be a devastating
and costly complication associated with TKA, with treatment
typically involving multiple surgical procedures and prolonged
courses of antibiotic therapy (Peel et al., 2013; Tande and Patel,
2014). MSSA causes approximately 45% of PJI cases, and is 2.5
times more likely to be associated with a PJI than MRSA (Guo et al.,
2017; Tande and Patel, 2014). Despite apparent antibiotic sensitivity
in vitro, the ability of MSSA strains to form biofilms on orthopaedic
implants in vivo continues to pose a significant challenge for
treatment of delayed- and late-onset PJI. Identifying alternate
strategies for treating PJI is currently a priority area of research, and
small animal models will continue to play a key role in translation of
new therapeutic approaches (Schwarz et al., 2019). While several rat
models of implant-associated S. aureus infection have been
described, clinical representation of modern TKA in terms of the
combination of orthopaedic materials used has been limited (Lin
et al., 2017; Reizner et al., 2014; Schindeler et al., 2018; Stadlinger
et al., 2013). Previously described models have used intramedullary
insertion of stainless steel implants into the distal femur or the
proximal tibia (Edelstein et al., 2017; Lucke et al., 2003), or extra-
articular insertion of screws or pins into bone (Stadlinger et al., 2013).
We describe the surgical technique for a novel rat model of knee
implant surgery based on materials currently used in TKA. Further,
using a previously characterised clinical MSSA strain to colonise
the femoral titanium implant prior to surgery, we successfully
progressed our uncomplicated surgical model to one representative of
delayed-onset PJI.
Additive manufacturing technology, or 3D printing, is increasingly
being applied to orthopaedics as a result of its capacity to blend
different materials such as titanium into diverse pore sizes and
thicknesses so as to mimic the porosity and stiffness of bone (Bravin
and Dietz, 2018). Newer generation TKA implants comprise 3D-
printed highly poroustitanium-coated components that exhibit a high
degree of osseointegration following cementless fixation (Bravin and
Dietz, 2018). UHXLPE is commonly used for covering articulating
surfaces due to its favourable wear rate (Dion et al., 2015; Le et al.,
2014). Recently, Carli et al. (Carli et al., 2018) described a mouse
model of implant-related osteomyelitis based on a press-fit titanium
tibial implant comprised of an articular baseplate and intramedullary
stem. Using intra-articular inoculation of S. aureus at the time of
surgery, the authors report the establishment of high-level, purulent
infection with elevated systemic inflammatory markers (Carli et al.,
2018). While this model offers the advantage of an implant with a
weight-bearing surface, it may not accurately represent the clinical
features of delayed-onset, low-level PJI for which blood cultures
remain negative and systemic inflammatory markers are often within
normal ranges (Beam and Osmon, 2018). To our knowledge, only
one study has used a combination of metal and high-density
polyethylene press-fit femoral and tibial implants for development of
a rat model of PJI (Søe et al., 2013). Using non-constrained knee
prostheses, Søe et al. (2013) demonstrated establishment of
osteomyelitis in rats following direct seeding of >10
3
CFU S.
aureus into both the femoral and tibial defects at the time of knee
surgery. Semi-quantitative methods were subsequently used to infer
implant stability and bacterial persistence in this non-constrained
model (Søe et al., 2013). Notably, implant stability and long-term
success of TKA is dependent on component fixation that can either be
cemented (constrained) or cementless (non-constrained) (Dalury,
2016; Vertullo et al., 2018). Antibiotic-laden bone cement was
introduced in the 1970s for infection prophylaxis in arthroplasty, with
gentamicin one of the most commonly incorporated antibiotics
(Hinarejos et al., 2015). The distinction of our model is the use of a
hybrid fixation technique, making it more akin to the materials and
techniques currently used in TKA (Dion et al., 2015; Hinarejos et al.,
2015; Le et al., 2014; Lin et al., 2017) and the establishment and
persistence of a low-level PJI that is localised to the operated knee.
In the absence of infection, animals in the current study recovered
without complication and returned to full weight-bearing within the
first week of knee implant surgery. Although plasma CRP levels
were higher at day 28 post surgery in control animals than at
baseline, these levels remained within reported normal ranges for
CRP in rats (de Beer et al., 1982). Further, haematology parameters,
including leucocyte number, had returned to baseline levels by
day 28 post surgery, with no additional clinical, pathological or
histological evidence of inflammation in the operated limb of
control animals. Importantly, stability of both the femoral and tibial
implants, and bone ingrowth around the press-fit porous titanium
implant and cement mantle surrounding the UHXLPE implant was
evident within 4 weeks of surgery in the absence of infection in
control animals.
In the current study, pre-seeding of femoral titanium implants
with MSSA was sufficient to establish a persistent, non-lethal and
localised infection in 100% of animals. While some systemic
inflammatory markers (CRP, % PMN) were significantly higher in
infected animals compared to controls in the first week following
surgery, levels returned and were comparable to controls for the
remainder of the 4-week experimental period. This is consistent
with previous animal models of implant-associated osteomyelitis
(Carli et al., 2018; Lucke et al., 2003; Rissing et al., 1985) and the
Table 2. Microbiological results of blood, tissue and implant cultures
No.
positive
Tissue
weight (g)
Log
10
CFU
CFU/100 mg
tissue
Blood
Control 0/5 - - -
Infected 0/8 - - -
Spleen
Control 0/5 0.89±0.1 - -
Infected 0/8 0.96±0.3 - -
Liver
Control 0/5 14.82±1.1 - -
Infected 0/8 13.92±2.0 - -
Total joint; bone and tissue
Control 0/5 2.67±0.7 - -
Infected 8/8 3.21±0.6 3.3±1.0 410±746 (3–2173)
Femur
Control 0/5 1.13±0.3 - -
Infected 8/8 1.34±0.2 2.67±0.8 123±175 (5–490)
Tibia
Control 0/5 1.23±0.4 - -
Infected 5/8 1.52±0.3 1.96±2.1 690±1384 (0–4005)
Patella
^
Control 0/5 0.31±0.03 - -
Infected 5/8 0.35±0.1 0.9±1.1 48±98 (0–273)
Draining lymph nodes
#
Control 0/5 0.24±0.1 - -
Infected 3/8 0.46±0.2 0.61±1.0 14±31 (0–87)
Titanium implant
Control 0/5 - - -
Infected 8/8 - 3.3±1.0 -
UHXLPE implant
Control 0/5 - - -
Infected 1/8 - 0.09±0.2 -
^Patella includes infra and supra-patella tendon and surrounding synovial
tissue.
#
, inguinal and popliteal lymph nodes of right (operated) hind limb. Data
show mean±s.e.m.
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clinical features of delayed-onset PJI, where serum biomarkers have
proven unreliable for diagnosis (Beam and Osmon, 2018). Similar
to the clinical manifestations of delayed-onset PJI (Beam and
Osmon, 2018), overt signs of persistent infection in animals in
the current study included a sustained reduction in post-surgical
body weight and delayed return to full weight-bearing on the
operated limb.
Localisation of MSSA infection to the implanted knee was
confirmed in the current study by quantitative microbiological,
immunological and radiographic changes. Culture of peri-prosthetic
and sonicate fluid remains the gold standard for diagnostic
confirmation of PJI (Beam and Osmon, 2018; Schwarz et al.,
2019; Tan et al., 2018). Despite the use of antibiotic-laden bone
cement, bacteria were recovered from all excised titanium implants
and peri-prosthetic tissue of the femur, and from tibia and patella,
demonstrating spread of MSSA to adjacent tissues within the joint.
Although there was inter-individual variability in the distribution of
bacteria within joint tissue, the total number of MSSA recovered
from implanted joints at 4 weeks post surgery was consistent
(∼2×10
3
CFU). Bone remodelling, metaphyseal osteolysis, fibrosis
and inflammatory cell infiltration was apparent surrounding the
press-fit titanium implant of infected animals at day 28 post surgery,
with a 2.4-fold decrease in peri-implant BV compared to uninfected
control animals. Consistent with this, levels of IL-1β, which
activates osteoclasts to increase bone resorption (Nguyen et al.,
1991), were significantly increased in the implanted knee of
infected compared to control animals.
Synovial rather than serum inflammatory markers consistently
demonstrate greater diagnostic sensitivity for PJI. Measurement of
synovial IL-6, CRP, alpha-defensin, leukocyte esterase and
calprotectin have been shown to improve diagnostic accuracy in
the workup for PJI (Beam and Osmon, 2018; Wouthuyzen-Bakker
et al., 2018; Xie et al., 2017). Nevertheless, published cut-off points
for these biomarkers vary widely for PJI diagnosis. The reported
cut-off values for synovial fluid IL-6 and CRP, for example, range
from 359.3 to 30,750 ng/l, and 3.65 to 12.2 mg/l, respectively, in
Fig. 5. Inflammatory chemokines and cytokines in joint tissue of implanted knees from control (n=5) and infected (n=10) animals at day 28 post
surgery. Data show mean±s.e.m. *P<0.05, Mann–Whitney test.
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patients with PJI following knee or hip arthroplasty (Gallo et al.,
2018). Synovial fluid was not measured in the current study.
However, in addition to elevated IL-1β, levels of calprotectin, IL-6
and MCP-1 tended to be higher in peri-implant tissue of infected
animals compared to controls. Calprotectin is an antimicrobial
protein that abundantly presents in the cytoplasm of neutrophils and
is released upon an encounter with pathogens. While neutrophils
predominate in the acute response to infection, their numbers at the
site of low-grade or chronic infection tend to be lower, with
lymphocytes and monocytes also contributing to the leukocyte
milieu in PJI (Josse et al., 2019). IL-6 is produced by stromal and
activated immune cells at sites of inflammation or infection and
plays a key role in regulating not only early neutrophil infiltration,
but also, together with monocyte chemoattractant protein 1 (MCP-
1), the mononuclear cell shift that occurs during prolonged
inflammation or chronic infection (Gabay, 2006). The minor
elevations observed in joint tissue inflammatory markers were
consistent with the mild-to-moderate mixed neutrophil, monocyte
and lymphocyte infiltration into synovial and peri-implant tissue in
knees from infected animals.
Fig. 6. Joint histopathology. (A,B)
Representative images of Goldner
Trichrome-stained, resin-embedded femur
with titanium implant from (A) control and
(B) infected animals at day 28 post surgery,
where mineralised bone matrix,
erythrocytes and non-mineralised osteoid
(new bone) are stained green, orange and
red, respectively. Focal areas of fibrosis
(arrows) and necrosis (asterisk) were
evident in peri-implant tissue of the infected
animals. (C–H) Representative histological
images of implanted knees from control
(C,E,G) and infected (D,F,H) animals at day
28 post surgery stained with Haematoxylin
and Eosin.Compared to control animals
(C), synovial hyperplasia and inflammatory
cell infiltration (arrows) was evident in joint
capsular tissue of the implanted knee of
infected animals (D) at day 28 post surgery,
with occasional cocci observed with Gram-
Twort staining (inset, arrowheads). Loss of
normal bone architecture, peri-implant
fibrosis and inflammatory cell infiltration
(arrows) were observed adjacent to the
titanium implant and in the periosteum
within the femur of infected animals (F,H)
compared to control animals (E,G).
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The major limitations of this study were the non-tribological
nature of the implants, the non-physiological method used for
inoculation and the relatively high numbers of S. aureus pre-seeded
to the titanium implants. The inoculation dose used in our study is
consistent with those reported for other rat PJI models using
different S. aureus strains (Reizner et al., 2014). Direct inoculation
of bacteria into the joint at the time of surgery was not used since
planktonic bacteria are more readily cleared by host immune
responses or potentially disseminate to surrounding tissues or
distant sites via haematogenous spread, thus increasing variability
in the infection site and the inoculating dose within the joint, and the
capacity for establishing infection (Arciola et al., 2018). Pre-seeding
of the titanium implants with ∼2×10
4
CFU provided a reliable
method for establishing a low-level, persistent and localised
infection in rats that is detectable 4 weeks after inoculation. We
emphasise that our purpose was to create a reliable experimental
model that reflects the key characteristics of delayed-onset PJI,
rather than its pathogenesis, per se. Importantly, our approach
consistently achieved the clinical features of delayed-onset PJI,
where osteomyelitis was initiated in the bone marrow adjacent to the
implant causing bone remodelling and local inflammatory changes
in the absence of systemic manifestations or clinically overt signs of
infection. Similar to the clinical scenario, no single biomarker or test
could reliably be used in lieu of microbiological culture to confirm
PJI in our experimental model. An additional limitation of the study
was that scanning electron microscopy (SEM) was not used to
visualise the structure of mature biofilms on explanted titanium
scaffolds at the study end as all samples were used for bacterial
quantification. Nonetheless, given bacterial loads were consistently
highest from titanium implant sonicates, it is likely that the
localised, persistent infection observed at day 28 post surgery was
associated with biofilm formation on the implant surface. The pull-
out strength of implants was also not assessed in the current study.
Rather, implants were carefully excised to improve accuracy of
bacterial load determination with osseointegration and implant
stability assessed by microCT analysis of peri-implant BV and
bone-implant contact.
In summary, we describe a surgical technique for knee implant
surgery in rats that is simple, reproducible, economical and recreates
the peri-prosthetic space akin to modern TKA. Further, key diagnostic
features of delayed-onset PJI (Beam and Osmon, 2018) were
demonstrated, with impaired return of joint function, sustained
weight loss after surgery, modest increases in inflammatory markers
within peri-implant tissue, evidence of peri-implant bone remodelling,
and positive culture from joint tissues and implant sonicates.
This model will serve as a useful and clinically relevant tool for
facilitating bench-to-bedside translation of new treatment approaches
for delayed-onset PJI caused by S. aureus biofilms.
MATERIALS AND METHODS
Bacterial biofilms
A previously described, a clinical isolate of S. aureus, ORI16_C02N, was
used in the current study (Morris et al., 2018). ORI16_C02N was isolated
from a patient who had presented with septic arthritis 3 years post TKA. This
strain has sensitivity to gentamicin, cefazolin and flucloxacillin.
ORI16_C02N is negative for panton-valentine leucocidin (pvl ) and the
plasmids, pUB110 and pT181, positive for collagen adhesion (cna) and
serine-aspartate repeat-containing protein E (sdrE), and belongs to clonal
complex (CC) 78.
In preliminary studies, BV SEM demonstrated the establishment of
mature ORI16_CO2N biofilms on the titanium implant surface within 24 h
of culture (Fig. S4). To more closely represent the early stages of biofilm
formation (adherence and colonisation), sterile titanium implants were
suspended in log phase cultures of ORI16_C02N (1.8×10
4
CFU, range 1.3–
2.9×10
4
CFU) for 12 h at 37°C, with a media change performed after 2 h to
remove non-adherent bacteria. Prior to implantation in rat femurs, biofilm-
coated titanium implants were rinsed twice in saline. Confirmation of mean
bacterial density within 12 h biofilms established on custom titanium
implants (n=5) was determined to be 1.2×10
6
CFU (range, 8.9×10
5
–
1.9×10
6
CFU) using sonication and enumeration of colonies on tryptic soy
agar (TSA) (Morris et al., 2018). Complete disruption of the biofilm was
confirmed with SEM (Fig. S4).
Animals
Twenty-week-old male Sprague-Dawley rats (350–450 g) were used. Animals
were individually caged, fed a standard pellet diet and provided water
ad libitum. At commencement of the 7-day acclimation period prior to surgery,
animals were randomised to control (n=12), or S. aureus-infected (n=13)
groups. Animalsin the infected group received titanium implantsthat had been
pre-coated with an S. aureus biofilm (described below). Control animals
received sterile titanium implants. Clinical signs including body weight,
temperature and weight-bearing activity were monitored daily throughout the
experimental period. Animals were sacrificed at 28 days post surgery with an
overdose of pentobarbital (100 mg/kg) for gross pathology, haematology,
microbiology, inflammatory, microCT and histological evaluation. All animal
experimental procedures were approved by the Institutional Animal Ethics
Committee (A2326).
Implants
3D-printed porous titanium implants were produced from Ti-6Al-4V, the
alloy used in human components, as previously described (Mazur et al.,
2016). The porous architecture was based on a cylindrical scaffold measuring
5 mm×1.6 mm, with a strut width of 205 µm and 70% porosity. Custom
cylindrical UHXLPE implants (5 mm×1.6 mm) were kindly produced and
provided by Enztec (Christchurch, New Zealand).
Surgical technique
All surgeries were performed within a sterile surgical field, using aseptic
techniques, sterile instruments, gowns, gloves and drapes. Knee implant
surgery was performed on rats under general anaesthetic with 5% isoflurane
(in 100% oxygen) during the induction phase and 2.5% isoflurane during
surgery, with animals breathing spontaneously (Fig. S5). Hair on the right
hind limb (ankle to abdomen) was clipped using sterile scissors, then
completely removed using hair removal cream (Veet
®
). The shaved area was
cleaned with chlorhexidine wash then swabbed liberally with 70% ethanol.
The right hind foot was swabbed liberally with alcoholic povidone-iodine
solution (10% w/v) using a sterile gauze and air-dried. Once dry, 3M™
Tegaderm™was applied around the foot. The shaved, right hind limb was
swabbed liberally with povidone-iodine and air-dried. A small fenestration
(2 cm×1.5 cm) was made in a large sterile drape (120 cm×120 cm) through
which the right knee was positioned so as to maintain a sterile surgical field.
Sterile IV3000
®
was applied over the flexed knee and fixed to the
surrounding drape.
The knee was opened with a medial parapatellar incision (12 mm) and the
patella dislocated laterally to expose the femoral condyles and tibial plateau.
A Microdremel and sterile titanium carbide drill bit (2 mm) was used to
create a defect in the proximal tibia. The UHXLPE implant was seated in a
small mantle of gentamicin-laden bone cement (Heraeus Palacos
®
R+G,
Zimmer Biomet, Sydney, Australia) which was mixed according to ratios
outlined in the manufacturer’s guidelines. Bone cement was deployed into
the tibial defect using a sterile, 3 ml syringe (Terumo, Sydney, Australia)
and the implant seated by gently tapping into place using sterile forceps.The
Microdremel and a second sterile titanium carbide drill bit (1.6 mm) were
used to create a defect between the distal medial and lateral femoral
condyles. Using forceps, the titanium implant was press-fit into the femoral
defect using gentle tapping. Following implantation, the patella was
repositioned and polydioxanone (PDS II) 5-0 (Ethicon, New Jersey, USA)
sutures were used to close the capsule. Skin was closed with monocryl 5-0
(Ethicon) using a continuous subcuticular technique and the surgical site
swabbed with povidone-iodine solution, then sprayed with OpSite™.
Immediately after skin closure and prior to recovery from anaesthesia,
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animals received pre-emptive analgesic consisting of a 0.05 mg/kg
subcutaneous injection of buprenorphine (Temgesic
®
) in a 1 ml bolus of
saline. Two additional doses of buprenorphine (0.05 mg/kg) were
administered at 6 and 12 h post surgery, with analgesic administered 8 to
12 times hourly thereafter, according to pain scores of individual animals.
Clinical signs including body weight, temperature and weight-bearing
activity were monitored daily throughout the experimental period.
Haematology and inflammatory assessments
Blood was collected under anaesthesia via the lateral tail vein during the
experimental period or via terminal cardiac puncture at day 28 post surgery.
Complete blood cell examination (CBE) was carried out using an automated
ACT Diff analyser (Beckman Coulter, Brea CA, USA). Blood samples were
centrifuged and plasma collected and stored at −80°C until further analysis.
CRP was measured in plasma using a Rat CRP ELISA (BD Biosciences,
North Ryde, Australia) according to the manufacturer’s protocols.
Calprotectin was measured in joint tissue homogenates using a Rat
Calprotectin ELISA (Cusabio, Houston, TX, USA) according to the
manufacturer’s protocols. Inflammatory chemokines and cytokines (MCP-
1, GRO/KC, MIP-2, TNF-α, IL-1β, IL-6, IL-12p70, IFN-γ, IL-4, IL-10) were
measured in plasma and joint tissue homogenates using Milliplex
®
Rat
Cytokine/Chemokine Magnetic Bead Panel (Abacus ALS, Meadowbrook,
Queensland) in combination with the Magpix
®
analyser (Luminex
Corporation, Austin, Texas, USA). Assays were carried out according to the
manufacturer’s instructions with samples measured in duplicate. Detection
ranges foranalytes were: 29.3–120,000 pg/ml for MCP-1; 14.6–60,000 pg/ml
for IFN-γand GRO/KC; 24.4–100,000 pg/ml for MIP-2; 2.4–10,000 pg/ml
for IL-1βand TNF-α;73.2–300,000 pg/ml for IL-6; 12.2–50,000 pg/ml
for IL-12p70, 4.9–20,000 pg/ml for IL-4; and 7.3–30,000 pg/ml for IL-10.
Assay sensitivities [minimum detectable concentration (pg/ml), intra-assay
precision (% CV) and inter-assay precision (% CV, n=11 assays)] for each
analyte were: MCP-1:9.0, 2.3, 9.2; GRO/KC: 19.7, 5.4, 7.7; MIP-2: 11.3, 2.9,
7.7; TNF-α: 1.9, 2.7, 10.8; IL-1β: 2.8, 3.6, 11.3; IL-6: 30.7, 2.3, 12.7;
IL-12p70: 3.3, 2.2, 7.8; IFN-γ: 6.2, 2.7, 12.4; IL-4: 3.1, 3.1, 10.7; and IL-10:
2.7, 3.8, 9.
Microbiological analyses
Bacterial loads were determined in blood, tissues and implants from animals
(control, n=5; infected, n=8) at day 28 post surgery. Briefly, liver, spleen,
popliteal and inguinal (draining) lymph nodes of the operated (right leg) and
non-operated (left leg) limb were dissected aseptically. The operated limb
was also removed at the hip and soft tissue was removed from the femur and
tibia and bone cutters used to cut the bone into small (<5 mm) pieces.
Titanium and UHXLPE implants were removed and sonicated as described
previously (Morris et al., 2018). The patella, associated tendons and
capsular tissue were processed separately from femur and tibia. Tissues were
homogenised using sterile stainless-steel beads (0.9–2.0 mm blend) and
Bullet Blender (NextAdvance, Troy, NY, USA). Blood and tissue
homogenates were serially diluted and cultured on TSA and mannitol salt
agar (MSA) overnight to enumerate bacteria.
Radiographic evaluation
Anterior–posterior (AP) and lateral x-ray images were taken of hind limbs
of animals (n=2) on day 7 post surgery to confirm implant positioning
(55–60kVp, 200 mA, 32 m s−1, 6.3 mAs; Shimadzu general unit and
digital detector plate, Canon CXDi-50G, Kyoto, Japan).
Micro-computerised tomography (µCT) scans
After sacrifice, hind limbs were removed from animals, with muscle and soft
tissue dissected from bone leaving the knee capsule intact (control, n=5;
infected, n=5). Specimens were fixed in 4% paraformaldehyde (48 h, 4°C)
prior to performing ex vivo microCT [Inveon PET-CT, Siemens, Bayswater,
Australia; 16.6 µm voxel size, 80 E(kVp), 200 µA, 0.36° rotation step]. Scan
images were reconstructed and bone parameters surrounding the press-fit,
femoral titanium implant were assessed using Materialise Mimics Innovation
Suite v20 (Materialise, Leuven, Belgium) with scan and reconstruction
parameters identical for all specimens.
Global thresholds were used to distinguish bone, soft tissue and the
titanium implant (bone, 1020–4142; titanium implant, 4142–20546).
Thresholds were based on visual inspection and were kept constant for all
scans. A one-voxel border surrounding the implant was excluded to account
for beam-hardening artefacts due to the metallic implant. Bone volume (%)
was determined within a cylindrical volume of interest (VOI) surrounding
the titanium implant using a series of mask dilations (55, 150 and 300 µm)
from the implant surface, then Boolean subtraction was used to determine
the intersect between bone within each sub-volume. Bone-implant contact
(BIC, %) was calculated as a percent ratio of the bone-implant intersect area
at a distance of 55 µm from the implant surface and the total surface area of
the titanium implant.
Histology
Following µCT scanning, representative knees were processed for resin-
embedded (control n=1, infected n=1) or routine histology (control n=3,
infected n=3). Briefly, hind limbs were dissected at the knee to separate the
femur and tibia without damaging the condylar surfaces. For resin histology,
bones were dehydrated using an ethanol series with increasing
concentration. Following dehydration, infiltration was conducted using a
mixture of ethanol and Technovit 9100 resin (Heraeus Kuzler, Hanau,
Germany), with an increasing ratio of resin over a period of 3 weeks.
Specimens were embedded using a UV embedding system and the
polymerised specimen block was longitudinally or transversely sectioned
at each implant centre using an EXAKT diamond cutting system (Kulzer
Exakt 300 CP). Blocks were attached to slides using an adhesive press
system and final slides were ground to a thickness ranging from the initial
200 µm to 48±5 µm using an Exakt grinding system (Kulzer Exakt 400 CS).
Goldner’s Trichrome staining was performed prior to mounting the sample
and the final slide images were scanned with a microscope (Axio M2
Imager, Carl Zeiss, Gottingen, Germany). For routine histology, intact knee
joints were decalcified in 14% EDTA, then bisected in an axial plane on the
lateral edge of the implant to enable sectioning of peri-implant tissue,
avoiding excessive tissue damage that would result from removal of the
titanium implant. Samples were processed and paraffin-embedded sections
(4 µm) stained with Haematoxylin and Eosin (H&E).
Statistics
Aprioripower analysis was conducted using the G-power
3
program to
determine appropriate sample size to reduce Type 1 errors (CFU in joint
tissue at day 28 post surgery; n=8). Statistical analyses were performed
using GraphPad Prism for Mac software (version 7). Data normality was
assessed using Shapiro-Wilks test, with Levene’s test used to determine
equality of variances. Independent samples t-tests were used for
between-groups comparison for normally distributed data. Changes in
haematology parameters for control and infected animals were compared
using two-way repeated measures ANOVA with Holm-Sidak post-hoc
analysis. Within-group differences were analysed with paired samples
t-tests or, where appropriate, repeated measures ANOVA with Dunnett’s
post-hoc analysis. Non-normally distributed data were compared using a
Mann–Whitney U-test or Kruskal–Wallis test with Dunn’s post-hoc
analysis. MILLIPLEX Analyst 5.1 software (Luminex Corporation,
Austin, Texas, USA) was used to determine cytokine and chemokine
concentrations with a 5-parametric logistic weighted curve fit. Results are
expressed as mean±standard deviation (s.d.), with significance set at
P<0.05.
Acknowledgements
We thank Mr Rob Wood (Stryker Orthopaedics), Professor Milan Brandt (RMIT) and
Mr Iain McMillan (Enztec Pty Ltd) for their kind provision of customised titanium and
UHXLPE implants. We are grateful to Mrs Lindy McEwen, Ms Regina Kirk, Dr
Genevieve Graw, Dr Ben Brandon, Dr Ivana-Aleksandra Jovanovic, Dr Tristan
Symonds and Mr Rhys Gillman for assistance with animal surgeries and sample
collection. We thank Ms Felicity Lawrence, Dr Darpan Shidid and Mr Rance Tino for
their assistance with resin-embedded histology and microCT scanning.
Competing interests
The authors declare no competing or financial interests.
11
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Author contributions
Conceptualization: J.L.M., A.G., M.W., K.H., P.M.; Methodology: J.L.M., H.L.L., A.G.,
M.W., K.H., P.M.; Formal analysis: J.L.M.; Investigation: J.L.M., H.L.L., P.M.;
Resources: J.L.M.; Data curation: J.L.M.; Writing - original draft: J.L.M.; Writing -
review & editing: J.L.M., H.L.L., A.G., M.W., K.H., P.M.; Project administration: J.L.M.;
Funding acquisition: J.L.M., A.G., M.W., K.H., P.M.
Funding
This research was supported by the Australian Orthopaedic Association, the
Townsville Hospital and Health Services Study, Education, Research and Training
Fund, and internal funds of the Orthopaedic Research Institute of Queensland and
James Cook University.
Supplementary information
Supplementary information available online at
http://bio.biologists.org/lookup/doi/10.1242/bio.045203.supplemental
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