Session 8a: Experimental
Session 8b: Sportstrauma

Torsdag den 23. oktober
13:00 – 14:30
Lokale: Stockholm/Copenhagen
Chairmen: Jan Duedal Rölfing / Martin Lind

73. The Göttingen Mini-Pig (GMP) as an Animal Model for Articular Cartilage Repair
Bjørn Christensen, Casper Foldager, Morten Olesen, Jan Rölfing, Steffen Ringgaard, Martin Lind
Ortopedic Research Laboratory, Aarhus University Hospital; Orthopedic Research Laboratory, Aarhus University Hospital; MR center, Aarhus University Hospital; Division of sports trauma, Aarhus University Hospital

Background: Several animal models are available for preclinical testing of cartilage repair, but a cost-effective and predictable large animal model is needed to bridge the gap between in vitro studies and clinical studies. Ideally, the animal model should allow for testing of clinically relevant treatments and the biological response should be reproducible and comparable to humans. This allows for a reliable translation of results to clinical studies.
Purpose / Aim of Study: This study aimed at verifying the GMP as a pre-clinical model for articular cartilage repair by testing several clinically available surgical treatment options and evaluation methods.
Materials and Methods: Thirteen fully mature GMPs were used. The GMPs received bilateral trochlear osteochondral drill-hole defects or chondral defects (Ø 6mm). The defects were treated with one of the following: Matrix-induced autologous chondrocyte implantation (MACI), microfracture (MFx), autologous- dual-tissue transplantation (ADTT), autologous bone graft, autologous cartilage chips. Empty chondral and osteochondral defects were used as controls. MRI and CT were performed 3 and 6 month, histology was performed at 6 month postoperative.
Findings / Results: The repair tissue varied in morphology from non-cartilaginous fibrous tissue to fibrocartilaginous tissue as seen on MRI, CT and histology at 6 month. The worst results were seen in the empty controls, the defects treated with autologous bone graft and defects treated with MFx. The best results were achieved with the MACI treatment.
Conclusions: The outcomes of the applied treatments were consistent with the outcomes in clinical studies. The GMP model was easy to handle, cost-effective and provided predictable outcome. Based on this study, we conclude that the GMP is a viable animal model for articular cartilage research.

74. Collagen type IV in Articular Cartilage Damage and Repair
Casper Bindzus Foldager, Wei Seong Toh, Bjørn Borsøe Christensen, Martin Lind, Andreas H. Gomoll, Myron Spector
Orthopaedic Research Lab, Aarhus University Hospital; Faculty of Dentistry, National University of Singapore; Sports Trauma Clinic, Aarhus University Hospital; Cartilage Repair Center, Brigham & Women's Hospital; VA Tissue Engineering Lab, Harvard Medical School

Background: Chondrocytes are surrounded by a thin layer of pericellular matrix (PCM). Collagen type IV (COL4) is present in the PCM of healthy chondrocytes. COL4 is composed of three of six different alpha-chains. Three of these alpha-chains contain angiogenesis inhibiting domains in alpha-1 (arresten), alpha-2 (canstatin), and alpha-3 (tumstatin), but their presence in cartilage is unknown.
Purpose / Aim of Study: To identify the COL4 alpha-chains in cartilage and describe its distribution of in cartilage damage and repair.
Materials and Methods: In vitro: Human chondrocytes from 6 patients were cultured in standard 21% oxygen and physiologic 1% oxygen for up to 6 days. Quantitative gene expression analysis (RT-qPCR) was performed to confirm the chondrogenic phenotype (Sox5, Sox6, Sox9) and to investigate the presence of the different COL4 alpha-chains 1-6. In vivo: Distribution of collagen type IV was investigated with immunohistochemistry in normal cartilage (human, n=5), damaged cartilage (human, n=5), clinically failed repair (human, n=11), cartilage repair with 6-month follow-up (minipig, n=11).
Findings / Results: COL4 expression in normal chondrocytes consisted of alpha chain 1 and 2. In normal cartilage COL4 was restricted to the PCM. Damaged cartilage contained significantly less COL4 in the PCM compared with normal cartilage. In minipigs with fibrous repair matrix COL4 was found in the extracellular matrix (ECM) and not the PCM. In fibrocartilage COL4 was also seen in the PCM and the interterritorial matrix. In hyaline repair tissue COL4 was restricted to the PCM. In failed repair COL4 was found in the PCM of 0-75% of the cells depending on tissue morphology and treatment.
Conclusions: COL4 in cartilage contain the angiogenesis inhibitors arresten and canstatin. The distribution of COL4 is changed in cartilage repair compared to normal cartilage.

75. Reduced force development in rat soleus muscle after exposure to Botulinum Neurotoxin A
Sofie Gjessing, Ole Rahbek, Juan Manuel Shiguetomi, Ole B. Nielsen, Bjarne Møller-Madsen
Dept. of Children's Orthopaedics, Aarhus University Hospital; Department of Biomedicine, Aarhus University

Background: A fast force development at the beginning of a muscle contraction is an important functional aspect of locomotion such as gait. In both human and rat motor function this can be achieved by firing double pulses (doublets) with an inter-pulse interval (IPI) as short as 1.6 ms. We hypothesize that Botulinum Neurotoxin A (BoNT) reduces the ability of the neuromuscular junction to transmit doublets and therefore also reduces their potentiating effect on force development.
Purpose / Aim of Study: We aimed to examine the effect of BoNT on force potentiation resulting from doublet stimulation in skeletal muscle.
Materials and Methods: Experiments were performed on isolated soleus muscle with intact motor nerve from juvenile Wistar rats that had been given intramuscular injections of BoNT 1-4 days prior to ex vivo examinations of contractile force.
Findings / Results: BoNT caused a progressive decline in nerve-stimulated tetanic force of the muscle preparations. In controls, nerve-stimulated doublets increased twitch force to ~200% of force of a single pulse when IPI was 2 ms and 230% for an interval of 4 ms. This potentiation was, however, attenuated by BoNT in a manner that depended on the reduction in nerve-stimulated force and the IPI. When tetanic force stimulated via the nerve was reduced to 0-39% of force produced by direct stimulation of the muscle fibres, force potentiation, resulting from nerve stimulation by doublets with an IPI of 2 ms, was significantly reduced (P<0.01, one-way ANOVA). No significant effects was observed at an IPI > 4 ms.
Conclusions: Treatment with BoNT in vivo reduced the ability of the neuromuscular junction to transmit high-frequency doublets in skeletal muscle. This indicates that treatment with BoNT may interfere with motor function during locomotion, which could compromise gait.

76. Inhibition of Micromotion induced Osteolysis in a Sheep Hip Arthroplasty Model
Thomas Jakobsen, Søren Kold , Juan Shiguetomi-Medina , Jørgen Baas, Kjeld Søballe, Ole Rahbek
Ortopædkir. Forskningslab., Aarhus Universitets Hospital; Ortopædkir. afd., Aalborg Universitets Hospital; Ortopædkir. Forskningslab, Aarhus Universitets Hospital; Ortopæd. Afd., Aarhus Universitets Hospital

Background: Aseptic loosening is the leading indication for revision surgery of a total hip replacement. It has previously been shown that early micromotion of a total hip replacement is associated with early aseptic loosening. One way to reduce the impact of the early micromotion on implant fixation could be with the use of bisphosphonates (BP). These compounds are strong inhibitors of bone resorption.
Purpose / Aim of Study: The aim of this study was to investigate whether local treatment with BP would reduce bone resorption around an experimental implant subjected to micromotion.
Materials and Methods: One micromotion implant were inserted into each medial femoral condyle in ten sheep. The micromotion device consists of an anchor bearing a PMMA implant and a PE plug. The 7.5 mm PMMA implant is placed exact-fit into a 7.5 mm bone cavity. During each gait cycle the PE plug will transfer load through the anchor and make the PMMA implant axially piston 0.5 mm. During surgery one of the femoral condyles were locally treated with 0.8 mg zoledronic acid. The other condyle served as control. Bone-implant specimens were harvested after 12 weeks of observation. Each specimen were embedded and vertically cut for histomorphometrical analysis.
Findings / Results: Histological evaluation showed a fibrous capsule around both the control and BP implants. Histomorphometrical analysis showed that 97% of the surface on both control and BP implants were covered by fibrous tissue (p=0.7). However, the BP was able to preserve bone in a 1 mm zone around the implants (66% bone volume fraction for BP vs. 57% bone volume fraction for control, p = 0.02).
Conclusions: This study indicates that local treatment with BP can not prevent the formation of a fibrous capsule around an implant subjected to micromotion. However, BP is able to reduce resorption of peri-prosthetic bone.

77. Sheep model reflecting glucocorticoid induced osteoporosis in postmenopausal women
Christina Møller Andreasen, Ming Ding, Søren Overgaard, Peter Bollen, Thomas Levin Andersen
Department of Ortopaedics & Traumatology O, Institute of Clinical Research, Odense University Hospital, University of Southern Denmark; Department of Orthopaedics & Traumatology O, Institute of Clinical Research, Odense University Hospital, University of Southern Denmark; Biomedicine Laboratory, University of Southern Denmark; Department of Clinical Cell Biology, Institute of Regional Health Services Research, Vejle-Lillebælt Hospital, University of Southern Denmark

Background: Sheep are often used as models for human osteoporosis, but it has not been verified whether the bone remodelling of sheep is comparable to human.
Purpose / Aim of Study: Here we investigate whether the bone loss in glucocorticoid treated ovariectomised sheep results from a similar bone remodelling defect as in osteoporotic postmenopausal women treated with glucocorticoids.
Materials and Methods: Ten sheep were ovariectomised and treated with prednisolone for 7 months. Ten untreated sheep served as control. The bone structure, selected histomorphometric parameters and the serum level of bone biomarkers were evaluated.
Findings / Results: Ovariectomy and glucocorticoid treatment induced a significant bone loss after 7 months. The extent of bone surfaces colonized by osteoclasts was unchanged, while the resorption marker CTX had a significant periodically elevation peaking after one month. The bone formation marker osteocalcin was consistently reduced after one week, and the extent of formative osteoid surfaces was almost undetectable after 7 months. The extent of reversal surfaces was significant increased after 1 month, covering almost 50% of the bone surfaces after 7 months. Most of these were arrested reversal surfaces without any neighbouring osteoclasts or osteoid surfaces, supporting that the bone formation and resorption were uncoupled during the reversal phase. The arrested reversal surfaces had a significantly reduced cell density and immunoreactivity for the osteoblastic markers runx2, osterix and SMA.
Conclusions: In sheep the bone loss results from an uncoupling of the bone formation and resorption during the reversal phase, as described in osteoporotic postmenopausal women treated with glucocorticoids, making it a relevant preclinical model for studying orthopaedic implant and biomaterial research under osteoporotic conditions.

78. Micromotion Induced Osteolysis in a Sheep Hip Arthroplasty Model
Thomas Jakobsen, Søren Kold , Jørgen Baas , Kjeld Søballe, Ole Rahbek
Ortopædkir. Forskningslab., Aarhus Universitets Hospital; Ortopædkir. afd., Aalborg Universitets Hospital; Ortopædkir. Afd. , Aarhus Universitets Hospital; Ortopæd. Afd., Aarhus Universitets Hospital

Background: Aseptic implant loosening might occur when peri-prosthetic bone anchorage is replaced by a fibrous membrane. It could be that the first step in the osteolytic cascade is micromotion-induced formation of a fibrous membrane around the implant.
Purpose / Aim of Study: The aim of this study was to investigate whether implants subjected to micromotion would be able to induce bone resorption and formation of a fibrous membrane without the presence of wear-debris particles.
Materials and Methods: One micromotion implant (T12) were inserted into one of the medial femoral condyle in ten sheep. The micromotion device consists of an anchor bearing a PMMA implant and a PE plug. The 7.5 mm PMMA implant is placed exact-fit into a 7.5 mm bone cavity. During each gait cycle the PE plug will transfer load through the anchor and make the PMMA implant axially piston 0.5 mm. After 12 weeks of observation the bone specimens were harvested and a post- mortem control implant (T0) was inserted into the contra-lateral medial femoral condyle. Each specimen were embedded and cut for histomorphometrical analysis.
Findings / Results: Histomorphometrical evaluation showed that the T12 implant surface was covered by fibrous tissue (93% for T12 vs. 0% for T0, p <0.001). The control implants were covered by lamellar bone (91% for T0 vs. 4 % for T12, p < 0.001). No difference was found with respect to the volume fraction of lamellar bone in a 1 mm zone around the implants (57% for T12 vs. 48% for T0, p = 0.2).
Conclusions: This study indicates that micromotion alone is sufficient to induce osteolysis and a fibrous membrane around an implant.

79. Vancomycin is superior to Plectasin against Staphyloccus aureus periprosthetic osteomyelitis in rats.
Niels H. Søe, Nina Vendel-Jensen, Asger Lundorff-Jensen, Janne Koch, Steen Sejer Poulsen, Helle Krogh Johansen
Hand Section,Department of Orthopaedic, Gentofte University Hospital; Department of Anaesthesiology,Intensive 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, Denmark

Background: Commonly used antibiotics cannot always control S. aureus associated infections in orthopaedic implants
Purpose / Aim of Study: We investigated the ability of Vancomycin and Plectasin to eradicate S. aureus in a knee prosthesis model of osteomyelitis in rats. We compared Plectasin in a 20 and 40 mg concentration to Vancomycin 40 mg, all antibiotics were given i.p. daily. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.
Materials and Methods: Thirty Sprague-Dawley rats had prosthesis inserted and divided into three groups (N=10) Vancomycin including 2 controls and two Plectasin groups (N= 10) 20 mg and (N= 10) 40 mg including 2 controls in each groups. All groups were infected with S. aureus MN8,ica+103 in the tibia and the femur marrow before insertion of the prosthesis. Control rats were given NaCl i.p. After two weeks, the rats were sacrificed and all specimens were analysed.
Findings / Results: One rat in the 20 mg Plectasin group and four in the 40 mg Plectasin group died of anaphylactic shock (histamine release). The 20 and 40 mg Plectasin groups both showed a decrease of bacteria but it was not as efficiently eradicated as in the Vancomycin group.
Conclusions: Plectasin treatment against S. aureus osteomyelitis reduced the infection. However, Plectasin released histamine strongly after one day and some of the rats died especially in the 40 mg group. In contrast, Vancomycin reduced the infection significantly in almost of the parameters.

80. Human chondrocytes cultivated on modified polystyrene conserve their chondrogenic phenotype in vitro
Natasja Leth Jørgensen, Dang Le, Casper Foldager, Martin Lind, Helle Lysdahl
Orthopaedic Research Laboratory, Aarhus University Hospital; Interdisciplinary Nanoscience Center, iNANO, Aarhus University; Sports Trauma Clinic, Aarhus University Hospital

Background: Autologous articular chondrocytes tend to dedifferentiate under prolonged expansion culture ex vivo. Porous scaffolds have been widely used to guide cells and grow new tissue.
Purpose / Aim of Study: In this study, we investigated the influence of precipitant induced porosity augmentation (PIPA) modified polystyrene surface on human chondrocytes (HCs) cultured in vitro. We hypothesized, that culturing HCs on 2D PIPA modified surfaces would conserve their phenotype.
Materials and Methods: The modification of polystyrene was made by the technique precipitant induced porosity augmentation (PIPA). Polystyrene was immersed with 1,4 dioxane and ozone treated prior to cell seeding. HCs were enzymatically isolated from cartilage biopsies collected from the inter-condylar groove in the distal femur. Isolated chondrocytes were expanded to passage 1 (P1) in DMEM/F12 supplemented with 10% FCS, 5 ng/mL bFGF, 1 ng/mL TGFβ3, and 1:100 penicillin-streptomycin (P/S). Chondrocytes in P1 were then seeded on PIPA modified polystyrene surfaces or on traditional monolayer (on a plan polystyrene surface) with 10,000 cells/cm2 in DMEM/F12 supplemented with 10% FCS and 1:100 P/S. HCs were cultivated until P4 and samples were collected for quantitative RT-PCR at each passages P1-P4. Toluidine blue and collagen II stains were performed on P2 HCs cultivated in pellets, 1×106 cells/mL, for 28 days.
Findings / Results: The PIPA surface promoted chondrogenic differentiation of HCs compared with traditional monolayer culture evident by higher gene expression of COL II, and the differentiation indices COLII/COLI and AGG/COLI. HCs expanded on PIPA surfaces prior to pellet formation revealed a better chondrogenicity by more synthesis of proteoglycans and collagen II.
Conclusions: Cultivation of HCs on the PIPA modified surface seems to conserve the chondrogenic phenotype.

81. Tunnel malpositioning in knee ligament reconstructions Denmark 2005-12
Thomas Hansen, Kim Lyngby Mikkelsen, Michael Rindom Krogsgaard
Section for Sportstraumatology M51, Bispebjerg Hospital; , The Patient Compensation Association

Background: Suboptimal positioning of tunnels is stated as the reason for cruciate ligament revision in 27 % of 1.866 operations reported to the Danish Kneeligament Reconstruction Register (DKRR) 2005-13. It is also the most common reason for compensation from The Patient Compensation Association (PCA) to patients after knee ligament reconstruction.
Purpose / Aim of Study: To describe the characteristics of tunnel malpositioning in knee ligament reconstruction reported to PCA and to evaluate causes and possible means of prevention.
Materials and Methods: From PCA patients with a reported complication 2005-2013 after knee ligament reconstruction were identified, and details in cases of tunnel malpositioning were studied. This was combined with information about the number of operations/year for each clinic (< 10, 10-50 and >50), if it was a public or private clinic and technique for femoral tunnel positioning.
Findings / Results: 92 malpositions were accepted for compensation. Of 52 malpositions in femur, 38 were anterior and 12 were vertical. Of 29 malpositions in tibia, 9 were anterior, 7 posterior and 13 medial, some perforating the medial tibial plateau. Combined malposition in femur and tibia was seen in 11. The incidence of tunnel malpositioning was highest in clinics with < 10 reconstructions/year (2,26 %), medium in clinics with 10-50 reconstructions/year (0,69 %), and lowest in clinics with > 50/year (0,28 %) (p < 0,05). 73 femoral tunnels were placed by transtibial (TT) technique and 11 by anteromedial (AM) technique – about 1/3 of all operations were done with AM technique and 2/3 with TT.
Conclusions: If 27 % of revisions were caused by tunnel malpositioning, less than 21 % of these had been reported to PCA. AM technique was less frequently connected to malpositions than TT technique. Routine seemed to be the best prevention against tunnel malpositioning.

82. One-year follow-up in the Danish Knee-ligament Reconstruction Register (DKRR) can be increased to 90 %
Jonathan Bjerre, Peder Klement, Bo Sparsø, Michael Krogsgaard
Department of sports traumatology, Bispebjerg; Department of orthopedic surgery, Bispebjerg; Center of health, Region Hovedstaden

Background: : The 1-year follow-up examination after ACL-reconstruction is a key quality parameter in DKKR. However, in the year report 2013 follow-up was reported for only 49,2 % (for Region Hovedstaden (RH) 45,6 %), which is a serious problem for the validity of the register. It has not been investigated why the reported follow-up is so low. The standard of >60% is based on the assumption that these patients are mobile and less motivated for follow-up.
Purpose / Aim of Study: To evaluate: completeness of 1-year follow-up after ACL-reconstruction in 2012 in RH, reasons why it is incomplete, ways to increase completeness and whether the official standard of > 60 % is realistic.
Materials and Methods: Patients who were registered with an intervention classified as KNGE45 or KNGE46 in 2012 the databases of 8 public and private hospitals in RH were identified, and their hospital files were studied. For patients who had no one-year follow-up it was checked if they had been invited. Data were compared with extracts from the DKRR.
Findings / Results: Of 931 operated patients, 814 were registered in DKKR (87,4 %). The follow-up rate reported to DKRR was 34,9 % (range 0-68 %). The actual follow-up rate was 64 % (range 30 – 91 %). There were marked differences in the way follow-up was planned. Most important reasons for lacking follow-up were failure to report to DKRR, no standard strategy for follow-up, technical problems with DKRR and unclear courses due to other operations. Less than 10 % of patients refused to show up.
Conclusions: A reported follow-up rate >90% is realistic if patents are invited at a reasonable time before follow-up, if they are re-invited once in case they don’t show up and if hospitals report actual follow-ups. Technically, reporting should be easier and robust to IT evolution.

83. Normative profiles for hip strength and flexibility in elite footballers
Andrea Mosler, Kay Crossley, Kristian Thorborg, Adam Weir, Andreas Serner , Per Hölmich
Sports Groin Pain Center, Aspetar Orthopaedic and Sports Medicine Hospital, Doha, Qatar; , University of Queensland; Sports Orthopedic Research Center - Copenhagen, Arthroscopic Center Amager; Sports Groin Pain Center & SORC-C, Aspetar Orthopaedic and Sports Medicine Hospital, Doha, Qatar & Arthroscopic Ceneter Amager

Background: Normative profiles for hip/groin strength and flexibility have not been established in footballers.
Purpose / Aim of Study: The primary aim of this study was to determine these normative profiles.
Materials and Methods: 345 males (18-40) years, playing professional football in the Qatar Stars League were evaluated. Strength was measured using a hand held dynamometer with an eccentric break test in side-lying for hip adduction (ADD) and abduction (ABD), and squeeze test in supine with 45° hip flexion. Flexibility measures included; hip internal rotation (IR) in both 90° flexion and prone, hip ABD in side-lying and bent knee fall out (BKFO). Demographic information was collected and the effect on the profiles was analysed. Pain on each testing procedure was recorded.
Findings / Results: Normative values for strength were as follows (mean±SD); ADD non- dominant=3.2±0.6 Nm/kg, dominant=3.1±0.6 Nm/kg; ABD both legs= 2.4±0.4 Nm/kg; ADD:ABD ratios non- dominant=1.4±0.3, dominant=1.3±0.3; Squeeze test=3.2±0.8 N/kg. Flexibility; IR in flexion both legs=37±8°, IR in prone non- dominant=36±8°, dominant=37±8°, ABD in side lying both legs=47±8°, BKFO non- dominant= 13.5±4.1cm, dominant=13.7±4.1cm. Leg dominance and a past history of injury had no clinically relevant effect on the strength and flexibility profiles. However, pain on squeeze testing significantly reduced the score for that test (p≤0.05).
Conclusions: Clinicians can confidently compare the strength and flexibility profile with the non- injured leg when assessing a footballer with unilateral pain. Pain on testing resulted in lower squeeze values, but a past history of hip or groin injury did not affect these profiles. Future studies will examine the predictive value of these measures for injury.

84. Complications after kneeligament reconstruction in Denmark 2005-2012 as reported to The Patient Compensation Association (PCA)
Thomas Hansen, Kim Lyngby Mikkelsen, Michael Rindom Krogsgaard
Section for Sportstraumatology M51, Bispebjerg Hospital; , The Patient Compensation Association

Background: The number and distribution of complications to kneeligament reconstruction is widely unknown, as many reports of complications origin from centers with selected patient groups. The Danish Kneeligament Reconstruction Register only report some types of complications, and the follow-up rate is quite low.
Purpose / Aim of Study: To describe complications to kneeligament reconstruction in Denmark as reported to PCA, related to number of reconstructions.
Materials and Methods: From PCA patients with a claimed complication after kneeligament reconstruction 2005-2012 were identified, excluding reconstruction of patellar ligaments. The files for each patient were studied, the complications were grouped and combined with information about rejection or compensation.
Findings / Results: Total number of reconstructions was 22.321. From 493 claimed cases, 261 (=1,17 %) were recognized for a compensation (total 35.558.205 DKK = 136.238 DKK/complication = 1.593 DKK/reconstruction). The 8 most common complications (claimed/recognized/total compensation in DKK): Tunnel malpositioning (104/92/13.221.872), deep infection (97/66/5.771.059), nerve damage (59/39/8.328.102), unrecognized diagnosis – typical undiagnosed multiligament injury (23/22/1.878.326), pain (46/18/1.022.676), arthrofibrosis (22/6/4.957.405), instrument failure (19/12/1.357.566), thromboembolic events (9/8/631.138). The 10 most expensive (6 tunnel malpositions) complications cost 15,8 mio DKK. Only six of 66 patients with infection had not received prophylactic antibiotics. Complications were not significantly more common in private (1,5 % of registered operations in DKRR) than in public (1,2 %) clinics, with individual variations.
Conclusions: Tunnel malpositioning is the most common complication, whereas nerve damage and arthrofibrosis are the (relatively) most expensive complications. Thromboembolic complications were rare.