J Mater Sci: Mater Med (2015) 26:130
DOI 10.1007/s10856-015-5467-6
BIOCOMPATIBILITY STUDIES
Staphylococcal biofilm formation on the surface of three different
calcium phosphate bone grafts: a qualitative and quantitative
in vivo analysis
Ulrika Furustrand Tafin • Bertrand Betrisey •
Marc Bohner • Thomas Ilchmann • Andrej Trampuz
Martin Clauss
•
Received: 23 October 2014 / Accepted: 9 January 2015
Ó The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract Differences in physico-chemical characteristics
of bone grafts to fill bone defects have been demonstrated
to influence in vitro bacterial biofilm formation. Aim of the
study was to investigate in vivo staphylococcal biofilm
formation on different calcium phosphate bone substitutes.
A foreign-body guinea-pig infection model was used.
Teflon cages prefilled with b-tricalcium phosphate, calciumdeficient hydroxyapatite, or dicalcium phosphate (DCP)
scaffold were implanted subcutaneously. Scaffolds were
infected with 2 9 103 colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted
after 3, 24 or 72 h of biofilm formation. Quantitative and
qualitative biofilm analysis was performed by sonication
followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed
U. Furustrand Tafin B. Betrisey A. Trampuz M. Clauss
Infectious Diseases Service, Department of Internal Medicine,
University Hospital Lausanne (CHUV), Lausanne, Switzerland
U. Furustrand Tafin M. Clauss
Unit of Septic Surgery, Department of Surgery and
Anaesthesiology, University Hospital Lausanne (CHUV),
Lausanne, Switzerland
M. Bohner M. Clauss
RMS Foundation, Bettlach, Switzerland
T. Ilchmann M. Clauss (&)
Department for Orthopaedics and Trauma Surgery, Clinic for
Orthopaedics and Trauma Surgery, Kantonsspital Baselland
Liestal, Rheinstreet 26, 4410 Liestal, Switzerland
e-mail:
[email protected]
A. Trampuz
Department of Traumatology and Reconstructive Surgery
including Department of Orthopaedic Surgery, Charité
Universitätsmedizin Berlin, Berlin, Germany
increasing amounts of biofilm on the surface of all scaffolds
over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no
biofilm was detected by microcalorimetry on the DCP
scaffolds after 72 h of infection. However, when using a
higher S. epidermidis inoculum, increasing amounts of
biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal
in vivo biofilm formation was observed between the different materials tested. This study highlights the importance
of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.
1 Introduction
Infections associated with medical devices rarely occur, but
represent a devastating complication with high morbidity
and substantial costs [1]. Depending on the causing
microorganism and host factors, these infections are typically
caused by microorganisms growing in biofilms [1]. These
microorganisms live clustered together in a highly hydrated
extracellular matrix attached to a surface. Existence within a
biofilm represents a basic survival mechanism by which
microbes resist against external and internal environmental
factors, such as antimicrobial agents and host immune system [2]. Depletion of metabolic substances and/or waste
product accumulation in biofilms causes microbes to enter a
slow- or non-growing state. Therefore, biofilm microorganisms are up to 1000 times more resistant to growth-dependent
antimicrobial agents than their planktonic (free-living)
counterparts [2–4]. For artificial joints and fracture-fixation
devices the most common microorganisms causing infection
are staphylococci [5, 6]. For prosthetic joint infection treatment is highly standardized [7] and eradication of infection is
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often only possible by removal of the implant and long-term
antimicrobial treatment [8].
Bone transplantation is the most commonly performed
transplantation, performed about 10-times more often than
any other solid organ transplantation [9]. More than one
million patients per year need a bone grafting procedure to
repair a bone defect resulting from a trauma or a bone
disease [10–12]. It is expected that bone grafts will be
increasingly used in orthopaedic surgery to fill bone defects, and be used also as antimicrobial delivery systems
[13]. The use of autologous cancellous bone grafts transplanted as fresh bone grafts is regarded as the gold standard
[10, 14, 15]. However, several bone graft substitutes have
been proposed, such as fresh-frozen allogeneic cancellous
bone grafts [16, 17] and processed human or bovine cancellous bone grafts [18]. All these genuine bone grafts have
a comparable calcium phosphate (CaP) architecture [11]. In
the 1970s, various compositions of synthetic CaPs, such as
b-tricalcium phosphate (b-TCP) or hydroxyapatite (HA),
were proposed. Their importance and use have considerably increased over the past decades [19]. Besides differences in physico-chemical properties, resorption and
osseointegration, artificial bone grafts differ in vitro in case
of staphylococcal colonization and biofilm formation [11,
20]. As there is an increasing use of these bone substitutes,
infections associated with these devices may also increase.
While the ‘‘race to the surface’’ [6] as a multistep process
of initial bacterial adhesion and later biofilm formation is
well established for metal implants [6, 21–24] there is only
limited data on in vitro [25, 26] and in vivo [27–29] biofilm
formation on the surface of different CaP bone graft substitutes, mainly HA and TCP.
There are various methods for quantitative/qualitative
evaluation of biofilm formation like ‘‘live-dead-staining’’
[30], confocal laser scanning microscopy [31], fluorescence
microscopy [23, 25], electron microscopy (REM/SEM)
[22, 23, 32] or atomic force microscopy (AFM) [32]. All
methods need a special pre-treatment like staining (livedead-staining, CFSM) or carbon-sputtering (REM/SEM)
which hinder further biofilm investigation after quantification or might be impossible to assess on rough or 3D
porous structures (AFM). In contrast, analysing biofilm
formation on the surface of various porous materials by
means of sonication and microcalorimetry has been shown
to be a robust test not necessitating a pre-treatment of the
biofilm in vitro [11, 20, 33].
In a recent in vitro study, we investigated by sonication
and microcalorimetry biofilm formation on the surface of
three different but morphologically similar CaPs, b-TCP
(cyclOS), dicalcium phosphate (DCP) and calcium-deficient HA (CDHA). We were able to demonstrate a lower
amount of biofilm on the b-TCP, compared to the DCP and
the CDHA. As the in vitro setting is very different from the
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J Mater Sci: Mater Med (2015) 26:130
clinical situation we wanted, as a next step, to confirm our
findings in an in vivo setting. The aim of this study was to
investigate in vivo biofilm formation on the surface of three
well characterized CaP bone grafts [b-TCP (cyclOS), DCP,
CDHA], and to compare the results to the in vitro data [33].
2 Materials and methods
2.1 Bone grafts
Three different CaP bone grafts [b-TCP (cyclOS), DCP,
CDHA], with recently published physico-chemical characteristic [20] were used (Table 1). Samples were obtained
as sterilized cylinders (6.5 9 10 mm).
2.2 Study organism
Two S. aureus strains (ATCC 29213, methicillin-susceptible and ATCC 43300, methicillin-resistant) [34] and one
S. epidermidis strain RP62A (ATCC 35984, methicillinsusceptible) [33] were used. The strains were stored at
-70 °C using a cryovial bead preservation system (Microbank, Pro-Lab Diagnostics, Richmond Hill, Ontario,
Canada). For preparation of the inoculum, a single bead
was freshly grown on sheep blood agar overnight. Bacterial
inocula were prepared from discrete colonies resuspended
in sterile 0.9 % saline (NaCl) to a McFarland turbidity of
0.5 representing a bacterial concentration of *1.0 9 107
colony-forming units (CFU)/mL. The stock solution was
diluted 1:1000 for further experiments.
2.3 Animal model
An established foreign-body infection model in albino
guinea pigs was used [35, 36]. The guinea pigs were kept in
the Animal House of the University Hospital Lausanne and
animal experimentation guidelines according to the
regulations of Swiss veterinary law were followed. The
study protocol was approved by the Institutional Ethical
Committee. In brief, four sterile polytetrafluoroethylene
(Teflon) cages (32 mm 9 10 mm) perforated with 130
regularly spaced holes of 1 mm in diameter (Angst-Pfister
AG, Zurich, Switzerland) prefilled with one CaP scaffold
were subcutaneously implanted in the flanks of male albino
guinea pigs (Charles River, Sulzfeld, Germany) under
aseptic conditions. Animals weighing 550–600 g were
anesthetized with subcutaneous injection of ketamine
(20 mg/kg of body weight) and xylazine (4 mg/kg). Two
weeks after surgery and healing of the surgical wounds,
interstitial fluid accumulating in tissue cages was checked
for sterility. Contaminated cages were excluded from further experiments. Experiments were performed in two
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Table 1 Summary of the physico-chemical properties of the samples used in the present study
Specific
surface area
(m2/g)
Macropore
diameter
(mm)
Apparent
density (g/
cm3)
Porosity
(%)
Porositya
(%)
Porosity accessible by
bacteria ([1.5 lm)a
(%)
b-TCP
[99 %b
(cyclOS)
0.84 ± 0.15
0.26 ± 0.07
0.88 ± 0.03
71.1 ± 1.0
70 ± 3
59 ± 3
17 ± 3
DCP
93 % DCP, 6 %
a-TCP, 1 %
DCPD
4.04 ± 0.35
0.37 ± 0.08
1.17 ± 0.04
60.0 ± 1.4
46 ± 2
37 ± 2
27 ± 7
CDHA
98 % HA, 2 %
DCP
43.6 ± 0.4
0.53 ± 0.13
0.53 ± 0.01
82.0 ± 0.3
69 ± 4
27 ± 9
0.23 ± 0.18
Materials
Compositions
da50 (lm)
Crystalline composition (Rietveld refinement analysis of the XRD data), specific surface area (SSA), macropore diameter, apparent density,
porosity, median pore size (d50) and porosity accessible by bacteria ([1.5 lm) in mean and standard deviation (from [20])
a
Determined by mercury porosimetry
b
Crystallite size 103 ± 12 nm (±1 St Dev)
animals in parallel carrying the same CaP scaffold in all
four tissue cages (i.e., eight replicates per material). On day
0, three out of four tissue cages/animal were infected by
inoculating 2 9 103 CFU/cage of either S. aureus ATCC
29213 (MSSA), S. aureus ATCC 43300 (MRSA) or
S. epidermidis RP62A ATCC 35984 (MSSE) with a sterile
syringe. The fourth uninfected cage served as negative
control. Animals were infected for 3, 24 and 72 h, respectively, according to an established in vitro setting [11].
Afterwards animals were killed by toxic CO2 and CaP
samples with the surrounding cage were harvested in the
animal house after disinfection of the skin and stored in
50 mL Falcon tubes prefilled with 5 mL 1 % of phosphate
buffered saline (PBS) for biofilm analysis (see hereafter).
2.4 Biofilm analysis
Biofilm analysis was performed under laminar flow and
adapted from our recently published procedure [33] including three steps (i) harvesting of the scaffolds and
washing procedure, (ii) sonication and (iii) a final microcalorimetric analysis.
CaP scaffolds to have a flush through the scaffold. Both the
glass pipette and the Pasteur pipette were changed after
processing one scaffold to avoid contamination from one
sample to another.
2.4.2 Sonication procedure
After washing, samples were transferred to new 50 mLFalcon tube containing 5 mL PBS, gently shaken for 10 s,
sonicated at 40 kHz for 1 min in a bath tub sonicator
(BactoSonic, Bandelin, Germany) and shaken again for
10 s. The dislodged biofilm (sonication fluid) was transferred to a 2 mL Eppendorf tube and CaP bone grafts were
stored for microcalorimetry (see hereafter).
Sonication fluid was serially diluted in Eppendorf tubes
and aliquots of 100 lL were plated on sheep blood agar and
incubated at 37 °C aerobically for 24 h. Bacterial counts
were enumerated and expressed as CFU/sample. Plates
were rated countable between 1 and 500 CFU/plate and
examined for variations in colony morphology (colour,
size) and contaminations.
2.4.3 Microcalorimetry protocol
2.4.1 Harvesting of the scaffolds and washing procedure
After harvesting of the scaffolds further processing was
done under laminar flow in the microbiology laboratory.
CaP scaffolds were transferred to a new 50 mL-Falcon tube
(prefilled with 5 mL PBS) with a sterile forceps after
peeling of the surrounding soft tissue envelope (Fig. 1a, b).
They were carefully washed five times with 5 mL 1 % PBS
to remove planktonic bacteria. For washing the PBS was
poured in the Falcon tubes by placing a glass pipette on the
wall of the Falcon tubes, afterwards the Falcon tubes were
shaken cautiously by hand and in a final step the PBS was
aspirated by placing a Pasteur pipette atop one side of the
All microcalorimetry tests were performed using a
48-channel batch calorimeter (thermal activity monitor,
model 3102 TAM III; TA Instruments, New Castle, DE,
USA).
In more details, CaP samples were transferred into
sterile 4 mL calorimeter ampoules pre-filled with 1 mL of
tryptic soy broth, closed with a rubber cap and sealed by
manual crimping. Ampoules were sequentially introduced
into the microcalorimeter and remained 15 min in the
thermal equilibration position before lowering into the
measurement position. Heat flow was measured continuously after the signal stability was achieved throughout
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Fig. 1 a, b CaP scaffold with surrounding soft tissue envelope explanted from the cage (right corner) and after peeling off soft tissue
an 18 h-period and expressed as heat flow over time [in
microwatts (lW)]. The calorimetric time to detection
(TTD) was defined as the time from insertion of the ampoule into the calorimeter until the exponentially rising
heat flow signal exceeded 20 lW to distinguish microbial
heat production from the thermal background. TTD indirectly quantifies the amount of bacteria with a shorter TTD
representing a higher amount of bacteria. Data analysis was
performed by the manufacturer’s software (TAM Assistant;
TA Instruments) and Prism 5.0 (GraphPad Software,
La Jolla, CA).
2.5 Statistical calculations
To equalize variances in bacterial counts, data are presented as log10 CFU/sample. For statistical analysis oneway ANOVA with Tukey’s multiple comparison test was
performed using Prism 5.0 (GraphPad Software, La Jolla,
CA). A P value \0.05 was considered to be significant.
3 Results
During the experiments none of the animals showed systemic signs of infection (i.e., all infections remained local)
and all animals showed the expected weight gaining over
time representing animal welfare. Uninfected CaP scaffolds used as negative experimental controls remained
sterile throughout the experiment.
Staphylococcus aureus ATCC 29213 and ATCC 43300
formed an increasing amount of biofilm on the surface of all
scaffolds over time (Fig. 2a, b). For both S. aureus strains a
statistically significant (P \ 0.05) increase was observed
between 3 and 24 h, and 3 and 72 h of infection, respectively,
on the three materials. There was no significant further
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increase in amount of biofilm between 24 and 72 h of infection. When comparing the three materials, significantly
less biofilm of S. aureus ATCC 29213 was detected by microcalorimetry on cyclOS compared to DCP and CDHA after
72 h of infection (P \ 0.05). However, no statistical difference between the materials was observed by sonication
and viable count. For S. aureus ATCC 43300, there were no
statistical differences between the three materials at any time
point.
For S. epidermidis RP62A the results were less homogeneous and a decrease in biofilm amount over time was observed for DCP and cyclOS (Fig. 2c). Sonication (CFU/mL,
left panel) showed a heterogeneous picture with an increase
amount of biofilm on the CDHA and DCP scaffolds but a
decreasing amount on the cyclOS scaffolds (not statistically
significant) between 3 and 24 h after inoculation. Furthermore, 72 h after inoculation, sonication showed no biofilm
on the surface of the DCP and cyclOS scaffolds indicating a
clearing of the infection, whereas a stable amount of biofilm
was detected on the CDHA scaffolds. At all time points,
significantly less biofilm was found on DCP compared to
CDHA (P \ 0.05) by sonication and viable count. After 24
and 72 h of infection, significantly less biofilm was also
found on cyclOS compared to CDHA (P \ 0.05). Less
biofilm was found on DCP compared to cyclOS after 3 h of
infection (P \ 0.05). Microcalorimetry (right panel) showed
a stable amount of biofilm on the CDHA scaffolds over time.
For DCP there was a stable amount of biofilm between 3 and
24 h after inoculation but a clearing of the infection after
72 h incubation (TTD [18 h). Both findings were in concordance with results obtained by sonication. On the cyclOS
scaffolds there was a decrease in biofilm amount between 3
and 24 h (P \ 0.05) after inoculation, which was in concordance with sonication. 72 h after inoculation microcalorimetry showed less biofilm on cyclOS as compared
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Fig. 2 S. aureus ATCC 29213 (a), S. aureus ATCC 43300 (b) and S. epidermidis RP62A (c) bacterial counts in sonication fluid (left) compared
with microcalorimetry results (right). * P \ 0.05
to 3 h (P \ 0.05) but more biofilm as compared to 24 h after
inoculation (not statistically significant). As observed by
sonication and viable counts, less biofilm was observed on
DCP and cyclOS compared to CDHA after 24 and 72 h of
infection (P \ 0.05). In addition, less biofilm was observed
on cyclOS compared to DCP after 24 h of infection.
Additional experiments with S. epidermidis RP62A with
a higher initial inoculum (from 1 9 105 to 1 9 107 CFU)
were performed to investigate whether clearing of the infection with the DCP and cyclOS scaffolds was due to the
material or if the initial inoculum had been too low to
establish a stable biofilm infection. With the higher
inoculum, the infection remained stable on all scaffolds but
the amount of bacteria found on the scaffolds by sonication
varied between the materials (Fig. 3, left panel). By
sonication, no bacteria could be dislodged from three of
three scaffolds for DCP, and in two of three scaffolds for
CDHA and cyclOS after 72 h of infection. In contrast,
microcalorimetry showed the shortest TTD at 72 h for all
three tested materials (Fig. 3, right panel). When comparing the three different materials, no significant differences
in biofilm formation was observed at any time point.
4 Discussion
Staphylococcal foreign-body infection is a significant
complication for orthopaedic patients undergoing surgery,
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Fig. 3 Results from additional experiments with a higher infection inoculum of S. epidermidis
particularly with fracture fixation and arthroplasty. Given
the difficulty in studying S. aureus infections in human
subjects, animal models serve an integral role in exploring
the pathogenesis of these infections, and aid in determining
the efficacy of prophylactic and therapeutic treatments.
Animal models should mimic the clinical scenarios seen in
patients as closely as possible to permit the experimental
results to be translated to the corresponding clinical care.
There is no animal model which is defined as the gold
standard for the investigation of staphylococcal biofilm
formation but the course of a foreign-body infection in the
guinea pig model is similar to that observed in humans
[37], and thus the guinea-pig model might come closest to
such a definition. In contrast to mice and rats no spontaneous cure of infected implants occurs [45]. As we expected small differences between the materials, all
experiments were performed with a relatively small starting bacterial inoculum (2 9 103 CFU/cage) as compared
to other experiments using the same strains using inocula
of 104–107 CFU/cage [34, 38–40].
We obtained a stable infection for both S. aureus strains
on the surface of all CaP scaffolds. Interestingly the
amount of biofilm was always lower for the MRSA (ATCC
43300) as compared to the MSSA (ATCC 29213) strain.
Even though differences were small this observation might
represent the reduced ‘‘fitness’’ of the MRSA strain which
can also be seen in the clinical situation. As observed in our
in vitro study [20], less MSSA biofilm was observed on
cyclOS compared to DCP and CDHA. However, in the
in vivo setting this could only be observed by microcalorimetry after 72 h of infection.
Our results obtained with the low inoculum of S. epidermidis were conflicting, when compared to in vitro results showing reduced biofilm formation on cyclOS using
the same materials and methods [20]. In the in vivo setting
less biofilm was detected on both DCP and cyclOS in
comparison to CDHA, and in addition less biofilm was
dislodged from DCP compared to cyclOS. When using an
infection inoculum of 2 9 103, we observed a spontaneous
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clearing of the infection 72 h on after inoculation of the
DCP material (e.g., no biofilm could be detected by viable
counts or sonication). Widmer et al. [40] did not observe
any spontaneous cure of S. epidermidis infection with a
starting inoculum of 104 CFU/cage using the same animal
model. Thus it remains unclear whether a stable infection
could be established in the cage or whether DCP is resistant
to S. epidermidis biofilm formation with the low inoculum
used. With the higher inoculum, microcalorimetry showed
comparable amounts of biofilm formation on the surface of
all CaP scaffolds, suggesting that a stable infection cannot
be established using a low infection inoculum on the DCP
material. DCP is considered to be acidic compared to bTCP or CDHA because it contains HPO4 groups instead of
PO4 groups [41]. Once present in the body, DCP can
theoretically convert to CDHA or HA releasing acidic
components (phosphoric acid) which might interfere with
bacterial growth.
Furthermore sonication and viable count showed a
clearance of the infection on the DCP after 72 h of infection even with the higher inoculum. This, in comparison to
microcalorimetry, contradictory result could be explained
by the higher sensitivity of the microcalorimeter. Whereas
the sonication allows quantification of detached biofilm
bacteria through viable count, the microcalorimeter measures the bacterial presence on and within the scaffold
during 18 h in a rich culture media allowing detection of
small bacterial quantities as well as dormant bacteria.
In a recent in vitro study, we investigated by sonication
and microcalorimetry biofilm formation on the surface of
morphologically similar CaPs. We found that biofilm formation was comparable for CDHA and DCP, but lower for
cyclOS [20]. These in vitro results suggested that biofilm
formation was not influenced by a single physico-chemical
parameter alone but is a multi-step process influenced by
several factors in parallel. Adherence to the surface involves
nonspecific physical factors (e.g., surface tension, hydrophobicity, and electrostatic interaction) and specific
bacterial and host adhesins such as fibronectin. This initial
J Mater Sci: Mater Med (2015) 26:130
process is followed by biofilm formation, which is mediated
in part by the polysaccharide intercellular adhesion (ica)
encoded by the ica operon [42]. While in the in vitro setting
bacteria were added to the CaP scaffolds after 30 min of
incubation in human serum [20], the time between implantation of the CaP scaffold and bacterial inoculation in the
in vivo setting was 14 days. A 2-week long period is needed
in order to allow complete wound healing after surgery. The
wound healing is especially important for animal welfare but
also for avoiding contamination of the implants during manipulation of the animals. Due to this prolonged time period
protein adsorption on the surface of the CaP scaffolds was
significantly different between the in vitro and in vivo setting. Whereas biofilm formation in an in vitro setting only is
influenced by nonspecific physico-chemical factors, the
in vivo setting includes the interaction between bacteria and
adhesins, especially fibronectin, covering the implant surface. In other words, the physico-chemical differences are
enveloped leaving only the macroscopic texture of the CaP
scaffolds which is rather comparable [20] explaining the
minor experimental differences between the materials in vivo. In order to be closer to the in vitro setting, another animal
model using pre-infected implants, such as the rat model
presented by Monzon et al. [43], could have been used. In a
clinical situation, bone grafts may be infected either during
surgery or post-operative due to disturbed wound healing
[44]. With post-operative contamination tissue integration of
the bone graft has already started and the tissue-cage model
used in this study might be more representative for the
clinical problem [44]. Another limitation of the study was
that the animal model used did not include local factors
generated during bone integration as the CaP scaffolds were
implanted subcutaneously and not directly into the bone.
5 Conclusion
Whereas, significantly less mature MSSA in vivo biofilm
could be observed on cyclOS compared to CDHA and
DCP, no significant variation in MRSA in vivo biofilm
formation was observed between the different materials
tested. With a low inoculum of S. epidermidis we found
less biofilm on DCP and cyclOS compared to CDHA, and a
clearance of the infection on the DCP bone grafts was
observed which might be explained by the release of HPO4.
The experimental setting represents an in vivo postoperative contamination model suitable to study the raceto-the-surface. This study highlights the importance of
considering in vivo factors when investigating biofilm
formation of bone grafts.
Acknowledgments This study was supported by research Grants
from 3R Foundation (S124-10) and AO Foundation (S-10-8C). At the
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RMS Foundation we want to thank S. Grünefelder and P. Brotschi for
their help producing the scaffolds. Furthermore we have to thank S.
Gersbach (Kantonsspital Baselland Liestal) for her help with data
management and analysis and Elena Maiolo (CHUV Lausanne) for
discussion of the results.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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