Session 5: Experimental
Torsdag den 24. oktober
11:00 – 12:00
Chairmen: Casper Bindzus Foldager / Martin Lind
Niels H. Søe, Nina Vendel-Jensen, Asger Lundorff Jensen, Janne Koch, Steen Seier Poulsen, Helle Krogh Johansen
Hand Section,Department of Orthopaedic, Gentofte University Hospital; Department of Anaesthesiology,intensice care and operations, Gentofte University Hospital; Faculty of Health and Medical Sciences, University of Copenhagen; Biomedical Department, Panum Institute, University of Copenhagen; Department of Clinical Microbiology, Rigshospitalet
Background: Commonly used antibiotics e.g.
vancomycin cannot always control S.
aureus associated infections in
orthopaedic implants and we have
tested immunized groups against a
Purpose / Aim of Study: In a prior study, we used vaccination to
boost the immune system in a knee
prosthesis model of osteomyelitis in
rats to explore the effectiveness of the
immune response achieved in active
as well as passive immunization and
tested this against the antibiotic
Materials and Methods: Sixty Sprague-Dawley rats were
operated and divided into two active
immunization groups (N=14 ica+/12 ica-
) and two passive immunization groups
(N= 12 ica+/12 ica-). A fifth group had
vancomycin ( N= 10 ica+). The ica-
gene controls the biofilm production.
All groups were infected with S. aureus
MN8,103 in the tibia and the femur
marrow before insertion of the
prosthesis. Each of the immunized
groups was compared to a non-
immunised control group and a
vancomycin group where the rats
received the antibiotics daily. After two
weeks, the rats were sacrificed and all
specimens were analysed.
Findings / Results: The active immunization groups
showed a decrease of bacteria in the
group that was infected with the ica+
strain. In the passive immunization
groups there was a clear decrease of
infection . The vancomycin group only
had few bacteria in two specimens and
was superior to the immunized groups.
Conclusions: Active and passive immunization
against S. aureus osteomyelitis
reduced the infection. However,
immunization based on only single S.
aureus virulence determinant may
have less protective efficacy because
of the multifactorial nature of the
pathogenesis of Staphylococcal
infection. Vancomycin reduced the
infection significantly in nearly all
Morten Lykke Olesen, Martin Lind, Helle Lysdahl, Casper Bindzus Foldager
Orthopaedic Research Laboratory, Aarhus University Hospital; Sports Trauma Clinic, Aarhus University Hospital; Institute for Clinical Medicine, Aarhus University Hospital
Background: Numerous preparation methods are
available for platelet-rich plasma (PRP)
generation, but evidence defining the
optimum composition is lacking.
Purpose / Aim of Study: The purpose of this study was to investigate
the effects of PRP containing low and high
leukocyte concentrations on both
proliferation and the chondrogenecity of
human chondrocytes in vitro.
Materials and Methods: Two PRP groups were generated from
whole blood from 9 healthy donors (age 36-
58 years) using a two-step centrifugation
protocol: low leukocyte PRP (llPRP) and
standard PRP (sPRP). The PRP groups had
similar platelet concentration but low and
high leukocyte concentrations, respectively.
Human chondrocytes were isolated from
articular biopsies obtained from 3 patients
with healthy cartilage (age 21-41 years), and
cultured in monolayer for 7 days in either
control media (DMEM/F12 with 10% fetal
calf serum) or control media with llPRP or
sPRP of 1%, 5% or 10% v/v concentrations.
Proliferation was assessed using an XTT
assay. qRT-PCR was used to perform
quantitative gene expression analyses using
primers for collagen type I (Col1a1) and II
(Col2a1) and Sox9. Data were collected on
day 2 and 7, and evaluated using three-way
Findings / Results: llPRP group showed an average leukocyte
concentration of 0.04 x 109 WBC/L, the
sPRP group 10.33 x 109 WBC/L. We
observed a positive proliferative effect by
both PRP groups compared with the control
group (p<0.0001). sPRP group showed an
increased proliferation compared with the
llPRP group (p<0.05). Presence of
leukocytes did not affect the relative mRNA
expression of Sox9, Col2a1, or Col1a1 in
any of the formulations.
Conclusions: We conclude that a high absolute leukocyte
concentration in PRP increase chondrocyte
proliferation. Inclusion of leukocytes in PRP
showed no effect on chondrogenic gene
Ming Sun, Miao Wang, Muwan Chen, Frederik Dagnaes-Hansen, Michael Robert Horsman, Cody Eric Bünger
Orthopedic Research Laboratory , Aarhus University Hospital; Interdisciplinary Nanoscience Center (iNANO), Aarhus University; Department of Biomedicine, Aarhus University; Experimental Clinical Oncology Department, Aarhus University Hospital
Background: Up to 70% of the breast cancer patients have bone metastases at autopsy-based
studies report. Until now, as for the treatment of bone metastases, local therapies,
including radiation therapy and surgery, were performed mainly as palliative
therapies. To effectively prolong the survival period and increase life quality of
patients with breast cancer bone metastases, new strategy for treatment is
Purpose / Aim of Study: To analyze the release profile of DESCLAYMR scaffold and test the anticancer
effect of this scaffold loaded doxorubicin in breast cancer cell lines and nude mice
Materials and Methods: 1. Different concentration of doxorubicin loaded to optimize the drug release: the fluorescence intensity of
doxorubicin in the buffer solution from each time point was quantified with a multilabel counter.
2. Cytotoxicity test of doxorubicin released from Desclaymr scaffolds in breast cancer cells by XTT and
Methylene Blue assay
3. Anticancer effect of DESLAYMR loaded with doxorubicin was tested in nude mice with subcutaneous
tumorand bone metastasis induced by breast cancer cell line, tumor volumes were monitored twice a week by
caliper and bioluminescent images.
Findings / Results: 1. DESCLAYMR released up to 60% of the drug for up to 3 months in vitro.
2. In tumor cells, DESLAYMR loaded with doxorubicin had higher cell inhibitory rate compared with
control group and a sustained cell inhibitory ability up to 12 weeks.
3.Average tumor volumes in treatment group increased slower than in non-treatment group from 3
days to 14 days after treatment. metastasis rate is lower in treatment group than in non-treatment
Conclusions: DESCLAYMR scaffold loaded with doxorubicin has a sustained release in vitro up to 3 months and
tumor growth and metastasis inhibitory effect in vivo.
Mikkel Tøttrup, Hanne Birke Sørensen, Tore Forsingdal Hardlei, Kurt Fuursted, Kjeld Søballe
Ortopædkirurgisk Afdeling, Hospitalsenheden Horsens og Ortopædkirurgisk Forskning i Århus; Ortopædkirurgisk Forskning i Århus, Aarhus Universitetshospital; Klinisk Biokemisk Afdeling, Aarhus Universitetshospital; Afdeling for Mikrobiologi og Infektionskontrol, Statens Serum Institut; Ortopædkirurgisk Afdeling E, Aarhus Universitetshospital
Background: Determining penetration of antimicrobials into bone tissue remains a difficult
task. The most commonly used method is bone biopsy. Different
approaches have been used. However, most seem to share
methodological and practical limitations.
Purpose / Aim of Study: The aim of this study was to validate and apply microdialysis for
measuring cefuroxime concentrations in bone tissue.
Materials and Methods: Initially in vitro studies were conducted in order to determine in vitro recovery by gain and by
loss, appropriate flow rate and calibration concentrations. The experiments were repeated at
different temperatures to assess the impact of this variable.
The prerequisite that recovery remains constant over a relevant period of time were tested in
vivo in swine. In order to assess whether microdialysis in drill holes in cortical bone solely
reflects bone concentrations, an experimental study in pigs was conducted where one of two
symmetric drill holes in the tibia was sealed with bone wax.
All holes were drilled using a 2 mm drill. In all experiments CMA 63 catheters were used. Flow
rate was 2 µl/min and was produced by a CMA 107 precision pump. All samples were analyzed
with an UHPLC-method. Intra-cortical placement of the catheters was verified by CT.
Findings / Results: 2 µl/min was found to be an appropriate flow rate, producing in vitro recoveries of
approximately 40-45 %. Recovery by gain was comparable to recovery by loss. Increasing
temperature increased recovery.
In vivo, recovery was found to be constant over time. No significant difference in area
under the curve, maximum concentration nor time of maximum concentration could be
detected between sealed and non-sealed drill holes in cortical bone.
Conclusions: Microdialysis seems to be a reliable method for determination of the
concentration of cefuroxime in bone tissue.
Juan Manuel Shiguetomi-Medina, Jose Luis Ramirez-GL, Ole Rahbek, Hans Stødkilde-Jørgensen, Bjarne Møller-Madsen
Department of Children's Orthopaedics / Orthopaedics Research Laboratory, Aarhus University Hospital; Clinical Investigation Sciences, Autonomous University of San Luis Potosi, Mexico.; Departiment of Children's Orthopaedics, Aarhus University Hospital; The MR Research Center, Aarhus University Hospital; Departmen of Children's Orthopaedics, Aarhus University Hospital
Background: Water is present in all human body. Healthy
tissues encompass an intricate balance of
water inside cells and extracellular matrix.
Disease can cause this relation to be altered.
It has been published that MR technology is
able to measure water content, but no
quantitative method has been described
Purpose / Aim of Study: Development of a mathematical model to
measure the water content in tissue using
T1-values obtained from MR
Materials and Methods: T1 values were obtained from 45
samples from tissue-mimicking gelatin
with previously known water
concentrations. We analyzed the
samples in a 1.5 Tesla by calculating
absolute T1 values in real maps through
inverse angle phase inverse sequence
recuperation (11 inversion times, from
200 to 2200 msec) at 37(±0.5) °C.
Regions of interest were manually
delineated and the mean T1 value was
estimated using a T1-map analysis
software. The collected data was
modeled in a linear regression by fitting
the values in the equation Water Content
~ T1-value at 95% confidence level
Findings / Results: The model was found to be statistically
significant against a null model (p <
0.001). R2 value was 0.973, meaning
that 97.3% of the variation in Water
Content can be explained by the T1
value. We validated the method with 150
bootstrap repetitions to an R2 corrected
index of 0.9715.
If T1 Signal Intensity is increased by 1
unit, Water content is increased by
0.019% [95% CI: 0.00018 – 0.00020], p <
0.001. Water content in percentage can
be predicted with absolute T1 values by
the equation Water Content = (0.476 + T1
Signal Intensity * 0.000193) * 100
Conclusions: Water content can be calculated using
absolute MR T1 values from. This
technology allows quantification of disease
manifestations such as edema and offers
bases for new research. This experimental
model has to be validated for human tissue
Jan H. Duedal Rölfing, Anette Baatrup, Maik Stiehler, Jonas Jensen, Helle Lysdahl, Cody Bünger
Orthopaedic Research Laboratory, Aarhus University Hospital; Department of Orthopaedics and Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital Carl Gustav Carus at Technische Universität Dresden, Germany
Background: We have previously shown that EPO elicits
osteogenic and angiogenic potencies in vitro
and in vivo. However, the cellular
mechanisms in human mesenchymal
stromal cells (MSCs) remain unknown.
Purpose / Aim of Study: The aim of this study was to investigate the
presence of pleiotropic EPO receptor
EPOR/CD131 on the cell surface and to
determine the involvement of intracellular
pathways TOR Serine-Threonine Kinase
(mTOR), Janus Kinase 2 (JAK2) and PI3K.
Materials and Methods: MSCs from two donors were cultured at
3000 cells/cm2 and treated with 20 IU/ml
EPO for 24 hours. Flow cytometry and
confocal microscopy were the primary
outcome measurements for receptor
experiments. Mineralization assays,
Arsenazo and Alizarin Red, assessed
intracellular pathways after 10 and 14 days.
Specific pathway blockers were used:
rapamycin, AG490, LY294002 and
wortmannin. The positive control was 20
IU/ml EPO and proliferation medium served
as negative control. Results were
normalized to cell number and isotype
controls were employed. Statistics consisted
of one-way ANOVA and Fisher’s LSD post
hoc testing of the pathway inhibitors vs.
EPO group. The skewed flow data were
analyzed with Mann Whitney test.
Findings / Results: Both EPOR and CD131 receptors were
observed on the cell surface of MSCs. Co-
expression of EPOR and CD131 was
observed in nearly all cells (range 95.7-
99.5%). All pathway inhibitors diminished
mineralization ranging from 18 ±2% to 70
±9% relative to the EPO group (p<0.0001).
Hence, all three examined pathways
contribute to osteogenic differentiation.
Because rapamycin inhibition was most
pronounced (82 ±2%), mTOR pathway is
the chief contributor to osteogenic EPO
Conclusions: For the first time, cellular mechanisms of
EPO's osteogenic effect on MSCs are
described. The non-hematologic
EPOR/CD131 receptor triggers mTOR,
JAK2 and PI3K signaling.
Morten Lykke Olesen, Martin Lind, Helle Lysdahl, Casper Bindzus Foldager
Orthopaedic Research Laboratory, Aarhus University Hospital; Sports Trauma Clinic, Aarhus University Hospital; Institute of Clinical Medicine, Aarhus University Hospital
Background: The majority of studies regarding platelet-
rich plasma (PRP) uses a simple two step
preparation protocol, but none has yet
considered how this approach alter white
blood cell (WBC;leucocytes) composition.
Purpose / Aim of Study: The purpose of this study was to investigate
how a two-step PRP preparation protocol
affects WBC composition.
Materials and Methods: Blood samples were obtained from 4 healthy
donors (age 26-31 years). Four different
plasma supernatant suspensions were
prepared from whole blood (WB) after the
first separation spin (250g, 10min) of a two-
step centrifugation protocol. With the line
separating the buffy coat layer (specific
gravity = 1.06) and the red blood cell (RBC)
layer (specific gravity = 1.09) serving as
reference line for the volume of the standard
suspension (sPRP), three larger volumes at
2.5mm, 5mm, and 7.5mm below the
standard reference line were collected.
Complete blood count analyses from each
sample were performed with an automated
hematology analyzer. All quantitative
measurements were described using
summary statistics (n, mean, standard
Findings / Results: The leukocyte concentration in WB ranged
from 3.69-7.52x109 WBC/L (mean
5.03±1.72x109 WBC/L) containing 55.5%
±5.1% neutrophils, 32.8%±3.6%
lymphocytes, and 8.4%±0.9% monocytes.
In sPRP the leukocyte pool contained 5.7%
±2.1% neutrophils (9.7-fold decrease
compared with WB), while the lymphocytes
represented 81.1%±6,7% (2.5-fold
increase). Below the standard reference line
the leukocyte pool gradually resembled WB,
but with increasing RBC concentrations.
Conclusions: The separation spin changed the
composition of the leukocyte pool in the
plasma supernatant. We showed that in
order to avoid RBC contamination in an
often-used PRP preparation protocol the
leukocyte composition changes from
predominantly neutrophils in WB to
lymphocytes in PRP.
Mette Sørensen, Jørgen Baas, Jeppe Barckman, Joan E. Bechtold, Kjeld Søballe
Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Aarhus University Hospital; Minneapolis Medical Research Foundations, University of Minnesota, MN, USA
Background: Local delivery of augments to stimulate
bone formation at the bone-implant
interface is desirable.
Purpose / Aim of Study: We investigate two polymers coatings,
poly(D,L-lactide) (PDLLA) and poly(lactic-
co-glycolic acid) (PLGA), as local delivery
systems and their effect on early implant
Materials and Methods: 12 dogs each received 4 experimental
implants surrounded by a 1-mm gap, two
implants in each proximal humerus.
Implants were: untreated titanium, PDLLA,
PLGA, PLGA+1.0mg simvastatin.
Observation was 4 weeks. We performed
quantitative histomorphometry to assess
fractions of new bone and fibrous tissue.
Mechanical evaluation by push-out test.
Statistical analysis by ANOVA and paired t-
test for the histomorphometrical data and
by Friedman and Wilcoxon for the
mechanical data. We considered p-values
<0.05 statistically significant.
Findings / Results: Both polymer coatings resulted in
significant inferior mechanical implant
fixation compared to untreated titanium
implants -most pronounced in the PLGA
groups. Simvastatin did not counteract the
negative effect of the polymer.
We found significantly more new bone
formation on the surface of untreated
implants compared to the all coated
implants. The coated implants had
significantly increased on-growth of fibrous
Conclusions: The residues from the polymer degradation
may result in local acidosis impairing
deposition of hydroxyapatite and may
decrease bone mineralization. The coating
may also act as a simple barrier for bone
on-growth making osseointegration
impossible until the polymer has
We were not able to determine if
simvastatin has any positive or negative
effect on implant fixation.
This study suggests that PDLLA and PLGA
are not suited as local delivery vehicles to
the bone-implant interface. Caution is
warranted when choosing delivery vehicle
for this purpose.