Session 9: Experimental
Torsdag den 22. oktober
13:00 – 14:30
Lokale: Helsinki/Oslo
Chairmen: Jan Duedal Rolfing / Ming Ding
97. Autologous cartilage chips transplantation for chondral defects: Improved repair tissue compared with microfracture
Bjørn Borsøe Christensen, Morten Lykke Olesen, Martin Lind, Casper Bindzus Foldager
Department of orthopedic surgery, Orthopedic research laboratory, Aarhus University Hospital; Orthopedic research laboratory, Aarhus University Hospital; Department of orthopedic surgery, Aarhus University Hospital
Background: Repair of chondral injuries using autologous
cartilage chips (ACC) have recently demonstrated
clinical feasibility.
Purpose / Aim of Study: The aim of this study was to investigate in vivo
cartilage repair outcome of autologous cartilage
chips and compare it with that of microfracture
(MFx) in full thickness cartilage defects. In addition
we wanted to evaluate the viability of the implanted
cartilage chips 6 months post-operative.
Materials and Methods: Six Göttingen minipigs (GMP’s) received two 6 mm
chondral defects in each knee. The two treatment
groups were 1) Autologous cartilage chips
imbedded in fibrin glue (ACC) (n=12), and 2) MFx
(n=12). The GMP’s were euthanized after six
months and composition of repair tissue was
quantitatively determined using histomorphometry.
Evaluation of the metabolic state of the
chondrocytes in the cartilage chips at follow-up
(normal or degenerative) was done by
immunohistochemistry measurements of collagen
type IV and laminin in the pericellular matrix.
Findings / Results: Significantly more hyaline cartilage was found in the
ACC (17.1%) compared with MFx (2.9%) (p<0.01).
Furthermore, there was significantly less fibrous
tissue in ACC (23.8%) compared with MFx (41.1%)
(p<0.01). No significant difference in fibrocartilage
content was found (54.7% vs. 50.8%). All cartilage
chips in the ACC group had >80% positive staining
for collagen type IV and laminin suggesting that the
implanted cartilage chips are not undergoing
degeneration six months after implantation.
Conclusions: ACC transplantation demonstrated better cartilage
repair tissue than MFx. The integrity of the PCM in
the cartilage chips suggested that the cartilage
chips were not degenerating. Implantation of viable
hyaline cartilage chips can explain the higher
proportion of hyaline cartilage in the ACC group.
98. Effects of substitute coated with hyaluronic acid or poly-lactic acid on implant fixation in glucocorticoid-treated ovariectomised sheep
Christina M. Andreasen, Ming Ding, Thomas L. Andersen, Søren Overgaard
Department of Orthopaedic Surgery and Traumatology O, Odense University Hospital, University of Southern Denmark; Department of Clinical Cell Biology (KCB), Vejle Hospital, University of Southern Denmark
Background: With an enhanced incidence of osteoporotic
fractures among the elderly population, the
need for a large animal model reflecting the
situation in osteoporotic patients has
increased. Recently we established that
glucocorticoid (GC)–treated ovariectomised
sheep replicates the bone remodeling defect
and bone loss in osteoporotic patients,
making it a suitable implant fixation model to
investigate the performance of bone
substitutes.
Purpose / Aim of Study: To investigate the performance of
substitutes consisting of hydroxyapatite
(HA) and beta-tricalcium phosphate (βTCP)
coated with hyaluronic acid (HyA) or poly-
lactic acid (PDLLA) versus allograft on
implant fixation in aged sheep with OVX and
GC-induced bone loss.
Materials and Methods: Ten sheep had osteoporosis induced by
OVX and GC-treatment for 6.5 months, and
subsequently four plasma-sprayed titanium
implant were inserted into their femur
condyles. The insertion created a
circumferential gap of 2 mm, which were
filled with one of the bone substitutes:
allograft (control), HA/βTCP, HA/βTCP-HyA
or HA/βTCP-PDLLA. After 12 weeks, the
implant specimens were analyzed by push-
out test and histomorphometry.
Findings / Results: Histology showed that the substitute groups
all contained remnants of un-resorbed
substitutes. However, bone formation was
found in all four groups, and only the HA/
βTCP group (zone 2) showed a statistically
significant lower bone formation compared to
the allograft group. The push-out test
revealed a similar mechanical fixation
comparing the groups, and likewise no
statistically significance difference in
ingrowth was found.
Conclusions: With HA/βTCP-granules coated with PDLLA
or HyA showing an osteoconductive
potential and mechanical fixation similar to
allograft, the coating materials are
considered valuable for composite materials
– even in osteoporotic bone.
99. The efficacy of bone substitutes on implant fixation in sheep
Christina M. Andreasen, Susan S. Henriksen, Ming Ding, Naseem Theilgaard, Thomas L. Andersen, Søren Overgaard
Department of Orthopaedic Surgery and Traumatology O, Odense University Hospital, University of Southern Denmark; New Medical Devices Initiatives, Danish Technological Institute; Department of Clinical Cell Biology (KCB), Vejle-Lillebælt Hospital, University of Southern Denmark
Background: The use of autograft is associated with
increased donor site morbidity, whereas
allograft has the risk of disease
transmission, and a limited availability. To
overcome these problems, synthetic bone
substitutes consisting of hydroxyapatite
(HA) and beta-tricalcium phosphate (β-TCP)
are used. The performance of these
substitutes can be enhanced by
reinforcement with poly (D, L)-lactic acid
(PDLLA) or infiltration with hyaluronic acid
(HyA).
Purpose / Aim of Study: To investigate the efficacy of HA/βTCP
granules reinforced with PDLLA or infiltrated
with HyA on the implant fixation in sheep.
Materials and Methods: Plasma-sprayed titanium implants were
inserted into the femur condyles of eight
sheep giving a 2 mm circumferential gap.
The gap was filled with one of the four
materials: allograft as control, HA/βTCP, HA/
βTCP-PDLLA or HA/βTCP-HyA. After 12
weeks the bone implant specimens were
analyzed by µCT-scanning, push-out test
and histomorphometry.
Findings / Results: Histomorphometric evaluation revealed the
formation of new bone in all groups with
remnants of bone substitute in the three
substitute groups. The allograft group
showed a larger bone volume in zone 1 and
2 compared to the HA/βTCP-PDLLA and
HA/βTCP group, respectively. There was no
statistically significant difference in the
mechanical fixation and the bone ingrowth,
comparing the four groups. The µCT
scanning showed a statistically significant
lower bone volume fraction in the allograft
group compared to the HA/βTCP and the
HA/βTCP-HyA group, which may be
explained by the non-absorbed bone
substitute material, which cannot be
distinguished from bone by µCT.
Conclusions: HA/βTCP granules infiltrated with HyA or
reinforced with PDLLA had similar bone
ingrowth and mechanical fixation as those
with allograft, signifying that the bone
substitutes may be considered as
alternatives to allograft.
100. On route to in-house production of tissue engineered implants
Dang Le, Cody Bünger, Ming Sun, Chi-Chih Chang
Afd E, Aarhus Universitetshospital; Afd. E, Aarhus Universitetshospital; iNANO, Aarhus Universitet
Background: Clinical restoration of organ function
using tissue engineering and
personalized bioresorbable implants has
been "just around the corner" for over
15 years. Although the technology
readiness level is very high, the
availability of clinically relevant
tissue engineering solutions or
individualized is virtually non-existing.
Purpose / Aim of Study: The aim of this pilot study is to chart
out the processing envelope for 3D stem
cell printing with regards to cell
viability, printing reproducibility, and
contamination risk.
Materials and Methods: Primary human mesenchymal stem cells
were loaded into a hydrogel blend of
hyaluronic acid, sodium alginate, and
hydroxyethylcellulose at a concentration
of 2 mio cells/ml. The composition of
the gel represented a balance between
biocompatibility, crosslinkability, and
printability, respectively.
The gel was printed as square constructs
measuring 10x10x2 mm using a nScrypt
3dn300TE fused deposition machine in a
ISO class 5 modular cleanroom.
Following printing, the gels were
crosslinked in 10 mM CaCl2 mixed with
DMEM + 10% FBS. Antibiotics were not
used. The cell-gel constructs were
cultured for 7 and visualized with
confocal microscopy with live-dead stain.
Findings / Results: We developed a lean workflow comprising
cell loading, gel homogenization,
printing, and crosslinking that ensured
a short processing times, high printing
printing speeds, good viability and
avoidance of contamination.
Conclusions: With limited commercial interest in
personalized implants, it may be the
task of the public hospitals to develop
this therapeutically very promising
concept. We have demonstrated how this
may be feasible.
101. Educational offer – Status on the average Danish orthopedic department
Eske Brand, Marie Fridberg, Ulrik Kragegaard Knudsen, Kristoffer Weisskirchner Barfod
Orthopaedic surgery, Holbaek Hospital, Denmark; Orthopaedic surgery, Clinical Orthopaedic Research Hvidovre, Copenhagen University Hospital, Hvidovre, Denmark
Background: In Denmark, the education of young
orthopedic surgeons is competency driven
with no final examination at the end of
residency. There are no national or
international studies describing the
educational activity among orthopedic
residents.
Purpose / Aim of Study: To examine the educational offer during
residency in Danish orthopedic departments.
Materials and Methods: The study was performed as a
questionnaire based cross-sectional
study. All residents n = 163 received a
web-based questionnaire containing 15
questions within the areas of workload,
surgical procedures, outpatient clinic
and research. The study period was from
January 1st to April 30th 2014.
Residents with at least 3 active months
at a department were included. Residents
with less than 80% completion of the
questionnaire were excluded.
Findings / Results: 152 entries were registered. 27 did not
meet the inclusion criteria and 29 were
excluded, leaving 96 participants,
representing 22 of 26 departments, for
further analysis. The average number of
operative procedures as primary surgeon
was 16 (range 8;35) per month. 18 out of
22 (81%) departments offered the
possibility to participate in research
facilitated by the department. 79 of 96
(79%) residents worked overtime (average
18 hours a month (range 5;43). 38 of 96
(40%) worked for free (average 10 hours
a month (range 3;60) to increase the
amount of surgical procedures.
Conclusions: A large variation in the educational
offer was found among the Danish
orthopedic departments. Some residents
are exposed to 2-3 times as many
surgical procedures as others. Danish
residents operate considerable less than
their US counterparts, but time spent on
work is more efficient. Many work for
free indicating the residents feel they
lack operative experience.
102. Determining the tissue concentration of dicloxacillin using Microdialysis
Kristian Kraft Hansen, Lasse Enkebølle Rasmussen, Ole Skov, Flemming Nielsen, Tore Bjerregård Stage, Uffe Jørgensen
Orthopeadic Research unit, department of Orthopedic Surgery and Traumatology, Odense University Hospital, Institute of Clinical Research, University of Southern Denmark; Department of Orthopedic Surgery, Vejle Hospital; Clinical pharmacology, Institute of Public Health, University of Southern Denmark
Background: Postoperative infections with s. aureus occur in
Denmark, despite the use of perioperative
dicloxacillin. Whether the tissue concentration of
dicloxacillin reached is above the minimal inhibitory
concentration (MIC) for S. aureus is unknown.
Purpose / Aim of Study: To measure the actual tissue concentration of
dicloxacillin in human muscle and adipose tissue and
compare these to the plasma concentrations.
Materials and Methods: Six healthy male volunteers were recruited. A CMA63
Microdialysis (MD) catheter (Mdialysis, Stockholm,
Sweden) was placed in the subcutaneous tissue of
the abdomen and in a muscle on the thigh. The
volunteers were given 2 g dicloxacillin intravenously
over 5 minutes. In 10 min intervals for the following 6
hours samples from blood and MD fluid were
collected. Recovery was determined in vitro.
Unbound dicloxacillin was isolated from plasma using
filter plates. All samples were analyzed with High
Performance Liquid Chromatography.
Findings / Results: Average recovery was 73,7 % Maximum
concentrations were reached in muscle tissue after a
median of 0.5 hours and adipose tissue after 0.8
hours. The geometric mean ration (GMR) of AUC0-6h
for adipose tissue compared to plasma was 0.32
[0.15-0.71]. Concentrations were above MIC for 3.4
hours in adipose tissue and 4.1 hours in muscle
tissue.
Conclusions: Dicloxacillin should be administered at least 30
minutes prior to incision to ensure maximum tissue
concentrations at the onset of surgery. A second
dose should be given after 3.4 hours in case of long
surgery time. Since the dicloxacillin concentration
reached in the adipose tissue is lower than in plasma
it should be investigated whether this difference is
more prominent in adipose patients or patients with
impaired peripheral circulation.
103. Staphylococcus aureus Infection on Knee Implants, with Focus on Biofilm
Kristine Marie Jakobsen, Niels H. Søe, Britt Mejer, Janne Koch, Claus Sternberg, Helle Krogh Johansen
Department of Systems Biology, Technical University of Denmark; Hand Section, Department of Orthopaedic, Gentofte University Hospital; Hand Surgery, Department of Orthopaedic Surgery, Slagelse Sygehus; Department of Experimental Medicine, University of Copenhagen; Department of Clinical Microbiology, Rigshospitalet
Background: Commonly used antibiotics cannot always
control S. aureus-associated infections on
orthopaedic implants.
Purpose / Aim of Study: The overall aim was to construct fluorescent
clinical S. aureus strains that would enable
in vitro and in vivo investigation of the
biofilm formation in rats, which would
promote the understanding of the genetic
regulations involved in biofilm formation and
the pathology of S. aureus.
Materials and Methods: Fluorescent clinical S. aureus MN8 isolates
were constructed and subsequently used to
cause infection in teen Sprague-Dawley rats
that had undergone knee prosthesis
replacement. The study contained three
groups. Group 1 was infected with
fluorescent S. aureus MN8, ica+ 10^3 CFU
(N=4), group 2 was infected with fluorescent
S. aureus MN8, ica÷ 10^3 CFU (N=4), and
group 3 (control group) was given sterile
NaCl i.p (N=2). The rats were injected with
S. aureus or NaCl in the tibia and femur
marrow before the knee prosthesis was
inserted. The tissue and implants were
analysed 14 days after surgery.
An in vivo investigation of the biofilm
formation was performed in flow-chamber
biofilm systems. All biofilms were grown in
AB-media for four days and subsequently
analysed using a CLSM.
Findings / Results: X-ray and clinical analyses taken 7 days
after surgery showed signs of commencing
osteomyelitis, which had worsened 14 days
after surgery especially in the ica-positive
group. 14 days after surgery, the bacteria
were completely eradicated from the rats.
Conclusions: The ica-positive infections showed a slightly
higher occurrence of osteomyelitis and gave
a higher infection score compared to the
ica-negative infections. These results show
that the expression of the ica-operon might
increase the biofilm formation and thereby
also the osteomyelitis, which is in
accordance with the ica gene that is known
to induce biofilm formation.
104. 3D Printed Polycaprolactone Scaffolds Result in Foreign Body Reaction in an Articular Cartilage Repair Model in Minipigs
Morten Lykke Olesen, Casper Bindzus Foldager, Bjørn Borsøe Christensen, Jens Vinge Nygaard, Helle Lysdahl, Martin Lind
Orthopaedic Research Laboratory, Aarhus University Hospital; Department of Engineering, Aarhus University; Sports Trauma Clinic, Aarhus University Hospital
Background: Microfracture (MFx) treatment is considered
to be first-line treatment option for focal
cartilage defects. Adjuvant therapeutic
options, such as tissue engineered
scaffolds, may improve formation of
cartilaginous repair tissue.
Purpose / Aim of Study: To investigate the articular cartilage repair
potential of a novel nanostructured
polycaprolactone (PCL) scaffold in a
porcine chondral defect model.
Materials and Methods: The scaffolds were produced by rapid
prototyping of a PCL backbone structure.
The nanostructure was created by
thermal induced phase separation of a
tertiary solution injected into the PCL
backbone. Biocompability of the PCL
material was tested in vitro. Ten
skeletally mature Göttingen minipigs
received a full thickness chondral defect
(Ø=6mm) in the medial femoral trochlea
in both knee joints (n=20). The defects
were randomized into two groups: 1)
MFx treatment, and 2) MFx+scaffold
(Ø=6mm, h=0.8 mm). MRI scans were
performed at baseline and at 3 and 6
months follow-up. After 6 months, repair
tissue was evaluated using histological
analysis, O'Driscoll score, ICRS cartilage
repair assessment score and MRI
MOCART score.
Findings / Results: The results of the biocompability study
demonstrated no cytotoxic effects.
Complete regeneration of the cartilage was
not seen in any of the treated knees. The
MFx group scored significantly higher, than
the MFx+scaffold group in both the
O'Driscoll score (P<0.001), and the ICRS
score (p=0.03). Histologic evaluation of the
repair areas revealed foreign body giant
cells in the scaffold-treated groups. No
significant difference was found in the MRI
MOCART score at any of the time-points.
Conclusions: PCL scaffolds with a nano-porous tertiary
structure resulted in foreign body reaction
and significantly lower histological scores
when used as adjuvant therapy to MFx
treatment compared to MFx alone.
105. Substrate stiffness affects proliferation of human chondrocytes
Natasja Leth Jørgensen
Orthopaedic Research Laboratory , Aarhus University Hospital
Background: Chondrocyte-based cartilage repair require
a solid dependent expansion of
chondrocytes ex vivo. Previous studies
have shown that cells behave differently on
different solid substrates. Therefore,
substrate stiffness might be a tool to control
the behavior of chondrocytes.
Purpose / Aim of Study: We investigated the proliferation of
chondrocytes when cultured on different
substrates. We hypothesized, that substrate
stiffness influences proliferation of
chondrocytes.
Materials and Methods: Human chondrocytes were enzymatically
isolated from cartilage biopsies collected
from patients undergoing anterior cruciate
ligament reconstruction. Cells were seeded
with 2,500 cells/cm2 on 'rigid' polystyrene
(control; standard culture surface) or 'soft'
polydimethylsiloxane (PDMS; silicone
surface) coated with 0.5 μg/cm2 fibronectin
and cultured for 1, 4, 7, & 10 days.
Subsequently, cell viability and proliferation
were analyzed using XTT assay and
methylene blue staining, and cell cycle
analysis using propidium iodide.
Findings / Results: The viability of chondrocytes was
significantly decreased when cultured on
PDMS at day 4 & 7. This effect was
balanced at day 10. The proliferation was
significantly increased when human
chondrocytes were cultured on PDMS
compared with controls. Representative
images revealed diverse growth of the
chondrocytes cultured on the two
substrates. On the rigid substrate a colony-
based outgrowth was observed compared
with a single cell-based outgrowth on the
soft substrate. Preliminary cell cycle data
demonstrate more chondrocytes in S- &
G2/M-phase in the PDMS cultured
chondrocytes compared with the control.
Conclusions: Culturing chondrocytes on a soft substrate
increased the proliferative capacity. These
findings constitute further investigations
aiming at elucidating the role of a softer
culture substrate when expanding
chondrocytes.
106. Are Young Orthopedics self-satisfied Delusionists?
Per Hviid Gundtoft, Eske Brand, Kristoffer Barfod
Orthopedic Surgery, Kolding Sygehus; Clinical Orthopedic Research Hvidovre (CORH), Dept. of Orthopedic Surgery, Copenhagen University Hospital, Hvidovre
Background: It is a general opinion in the world of
medicine that orthopedic surgeons differ
from doctors of other specialties in
terms of intellect, strength and
self-confidence. While the former have
previously been refuted, so far no
studies have investigated orthopedics
surgeon’s self-confidence.
Purpose / Aim of Study: To examine young orthopedics
self-perceived skills and self-confidence.
Materials and Methods: Young Orthopedics in Denmark were asked
to fill out a questionnaire, which
consisted of the validated General
Self-Efficacy Scale (GSE) and questions
about their self-perceived surgical
skills. The participants GSE was
compared with result from a research
database encompassing data from more
than 17000 persons around the world.
Significant difference (p<0.05) was
estimated by the Wilcoxon rank-sum test.
Findings / Results: 112 young orthopedics completed the
questionnaire. 5 were excluded due to
incomplete answers. Young orthopedics
GSE score was 3.05, average GSE score in
the database was 2.94 (p=0.06).
96% of young orthopedics believed their
surgical talent was average or above
when compared with colleagues, and 45%
believed they were “more talented” or
“far more talented” than their
colleagues. 99% believed their surgical
skills would be rated “average” or above
by their colleagues and 46% believed
they would be rated “better” or “a lot
better” than average. 75% believed that
when assisting a senior surgeon the
patient would “sometimes” (59%), “often”
(14%) or “always” (2%) be better off if
they were the one doing the surgery.
Conclusions: Danish patients can rest assured that
they are operated by a self-confident
surgeon as young orthopedics have high
confidence in their surgical skills. In
general young orthopedics are not
significantly more self-confident than
the average population.
107. Effects of BMP-2 on Implant integration with Bisphosphonate Background – a Dose Response Study
Rasmus Cleemann, Mette Sørensen, Jørgen Baas, Joan Bechtold, Kjeld Søballe
Orthopedic Research Laboratory & Elective Surgery Centre, Aarhus University Hospital & Silkeborg Regional Hospital, Denmark; Orthopedic Research Laboaratory, Aarhus University Hospital, Denmark; Department of Orthopedics, Aarhus University Hospital, Denmark; Excelen Center for Bone and Joint Research and Education, Minneapolis Medical Research Foundation, Minneapolis, MN, USA
Background: Poor bone stock around arthroplasties can reduce early implant
fixation and threaten long-term implant survival. The trabecular bone
bed for the implant is rough with craters. Allograft has been shown
to augument early implant fixation, but still has an uneven bone-
implant interface. A bone anabolic stimulus with BMP-2 used with
catabolic control by systemic bisphosphonate (BP), could increase
new bone formation and protect bone, to improving early implant
fixation
Purpose / Aim of Study: We wished to investigate the dose-response
relationship on implant fixation in grafted and open
gap settings with three different doses of BMP-2
delivered from the implant surface, on a background
of BP (to control resorption of allograft and newly
formed bone)
Materials and Methods: Twelve dogs each received eight Ti-porous coated
implants. Two implants in each proximal humerus
and one in each distal femur condyle. Implants were
coated with one of three BMP-2 doses (240 μg,60
μg,15 μg) or control. The humerus implants had a
2.5 mm gap filled with allograft. The femur implants
had a 0.75 mm open gap. After 10 days, a single
dose of Zolendronate 0.1 mg/kg was administered
IV. Observation time was 4 weeks. Implants were
evaluated by mechanical push-out and
histomorphometrical analysis.
Findings / Results: Grafted gap: Control implants had better mechanical
fixation on all parameters (strength, energy &
stiffness) compared to BMP-2 coated implants
(Low:1.8 fold; Medium:3-fold; High: 4.4-4.8; all
p<0.05).
Open gap: Low dose BMP-2 had best fixation on all
parameters, compared to control and other BMP-2
doses (Control:2.2-3.4; Medium:2.2-2.2; High:2.6-
3.0; all p<0.05).
Conclusions: The mechanical data suggest that BMP-2 should be
used cautiously in relation to implants. Future
histomorphometric analysis will identify the tissue
composition of the bone-implant interface.
108. The significance of graft size for graft tunnel healing
Yeliz Jakobsen, Ines Willerslev Jørgensen, Ming Ding, Uffe Jørgensen
Department of orthopedic Surgery O, Odense University Hospital, University of southern Denmark
Background: In ligament reconstruction graft tunnel
healing is needed for a good outcome.
Traditional mechanical thinking
recommends the same size tunnel and graft
in order to fill the tunnel. This sometimes
makes passages of the graft difficult and
potentially harms the graft. However,
healing is determined by cell biology and
vascularisation, which is inhibited by
mechanical compression.
Purpose / Aim of Study: The purpose of this study was to investigate
the significance of graf size for graft tunnel
healing.
Materials and Methods: In a rat model, two tendon sizes ( 0.5mm
and 0.9 mm) was randomly inserted into
two tibial tunnels in the right leg of 8
Sprague Dawley rats. Slow drilling with a
conical drill (0.9mm) was performed in order
to avoid necrosis.
Bone healing was followed longitudinally
with 3D micro-CT. After healing histological
analysis were performed to study graft-
ingrowth.
A validated visual scoring system was used
to evaluate the amount of bone formation in
the tunnel.
Findings / Results: 7 of 8 rats could be followed to tunnel
healing. One rat was excluded due to
infection.
At the 4th 3D micro-CT scan (day 11)
healing was seen in all tunnels. Tendon-to-
bone ingrowth was confirmed by the
histological analysis in all cases. There was
no difference between the healing rate of
the two graft sizes.
Conclusions: In a non necrotic bone tunnel ingrowth can
be obtained without stuffing the tunnel with
the graft. Clinically this implicates that a
graft smaller than the tunnel can heal. This
should be tested further in clinical studies.