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.