Session 5: Experimental

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 vancomycin group.
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 peptide vancomycin.
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 parameters.

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 ANOVA analysis.
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 expression.

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 needed.
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 model.
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 group.
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 signaling.
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 error).
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 fixation.
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 tissue.
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 disappeared. 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.