Multidrug resistance protein 1 silencing in osteosarcoma and chondrosarcoma cell lines.

Background: The poor response of metastatic osteo- and chondrosarcomas to chemotherapy could be the result of multidrug resistance (MDR), which may be overcome through the use of small interfering RNA (siRNA). However, several methodologic questions remain unresolved. Aims: To test the toxicity of three commonly used siRNA transfection reagents and apply the least toxic reagent to investigate the siRNA-induced MDR1 mRNA knockdown. Methods: The toxicity of TransIT-TKO, Lipofectamine 2000, and X-tremeGENE siRNA transfection reagents was investigated on osteosarcoma (MG-63) and chondrosarcoma (SW1353) cell lines. The toxicity was measured at 4 and 24 hours using a MTT toxicity assay. The least toxic transfection reagent was applied to investigate the siRNA-induced MDR1 mRNA knockdown effect using qRT-PCR. Furthermore, five housekeeping genes were assessed in the BestKeeper software to obtain mRNA expression normalization. Results: Lipofectamine 2000 was the least toxic transfection reagent, reducing the cell viability only in chondrosarcoma 24 hours following exposure to the highest dose. In contrast, TransIT-TKO and X-tremeGENE transfection reagents displayed a significant reduction in cell viability in both chondrosarcoma after 4 hours and in osteosarcoma after 24 hours. Significant MDR1 mRNA silencing of over 80% was achieved in osteo- and chondrosarcoma using Lipofectamine at a final siRNA concentration of 25 nM. No significant dose response was observed in knockdown efficiency in either Lipofectamine or siRNA concentration. Conclusion: Lipofectamine 2000 was the least toxic transfection reagent in osteo- and chondrosarcoma. Successful siRNA-induced MDR1 mRNA silencing of over 80% was achieved.

Biomechanical evaluation of annulus fibrosus repair with scaffold and soft anchors in an ex vivo porcine model.

INTRODUCTION: Altered biomechanical properties, due to intervertebral disc (IVD) degeneration and missing nucleus fibrosus, could be thought as one of the reasons for the back pain many herniation patients experience after surgery. It has been suggested to repair annulus fibrosus (AF) to restore stability and allow nucleus pulposus (NP) replacement and furthermore prevent reherniation. The aim of this study was to evaluate a new method for closing a defect in AF for use in herniation surgery. METHODS: Our repair method combines a polycaprolactone (PCL) scaffold plugging herniation and soft anchors to secure the plug. Ex vivo biomechanical testing was carried out in nine porcine lumbar motion segments. Flexion-extension, lateral bending and rotation were repeated three times: first in healthy specimens, second with a full thickness circular defect applied, and third time with the specimens repaired. Finally push out tests were performed to check whether the plug would remain in. RESULTS: Tests showed that applying a defect to the AF increases the range of motion (ROM), neutral zone (NZ) and neutral zone stiffness (NZS). In flexion/extension it was found significant for ROM, NZ, and NZS. For lateral bending and rotation a significant increase in ROM occurred. After AF repair ROM, NZ and NZS were normalized. All plugs remained in the AF during push out test up until 4000 N, but NP was squeezed out through the pores of the scaffold. DISCUSSION: A defect in the AF changes the biomechanical properties in the motion segment, changes that point to instability. Repairing the defect with a PCL plug and soft anchors brought the biomechanical behavior back to native state. This concept is promising and might be a viable way to repair the IVD after surgery.

Aarhus Regenerative Orthopaedics Symposium (AROS).

The combination of modern interventional and preventive medicine has led to an epidemic of ageing. While this phenomenon is a positive consequence of an improved lifestyle and achievements in a society, the longer life expectancy is often accompanied by decline in quality of life due to musculoskeletal pain and disability. The Aarhus Regenerative Orthopaedics Symposium (AROS) 2015 was motivated by the need to address regenerative challenges in an ageing population by engaging clinicians, basic scientists, and engineers. In this position paper, we review our contemporary understanding of societal, patient-related, and basic science-related challenges in order to provide a reasoned roadmap for the future to deal with this compelling and urgent healthcare problem.

[Shewanella algae infection after surgical treatment of Haglund's heel and rupture of the Achilles tendon].

This is a case report of soft tissue infection with the marine bacterium Shewanella algae that is rare in Denmark. The patient was a 43-year-old male and he was treated surgically for Haglund's heel, a bony protrusion at the calcaneus. After clinical healing the patient suffered a rupture of the Achilles tendon, which was treated surgically as well. The post-operative healing process proved to be protracted with a number of surgical wound revisions being necessary. A microbiology culture showed the presence of S. algae and after proper antibiotic treatment the patient recovered quickly.

Pharmacokinetics of cefuroxime in porcine cortical and cancellous bone determined by microdialysis.

Traditionally, the pharmacokinetics of antimicrobials in bone have been investigated using bone biopsy specimens, but this approach suffers from considerable methodological limitations. Consequently, new methods are needed. The objectives of this study were to assess the feasibility of microdialysis (MD) for measuring cefuroxime in bone and to obtain pharmacokinetic profiles for the same drug in porcine cortical and cancellous bone. The measurements were conducted in bone wax sealed and unsealed drill holes in cortical bone and in drill holes in cancellous bone and in subcutaneous tissue. As a reference, the free and total plasma concentrations were also measured. The animals received a bolus of 1,500 mg cefuroxime over 30 min. No significant differences were found between the key pharmacokinetic parameters for sealed and unsealed drill holes in cortical bone. The mean ± standard error of the mean area under the concentration-time curve (AUC) values from 0 to 5 h were 6,013 ± 1,339, 3,222 ± 1086, 2,232 ± 635, and 952 ± 290 min · µg/ml for free plasma, subcutaneous tissue, cancellous bone, and cortical bone, respectively (P < 0.01, analysis of variance). The AUC for cortical bone was also significantly different from that for cancellous bone (P = 0.04). This heterogeneous tissue distribution was also reflected in other key pharmacokinetic parameters. This study validates MD as a suitable method for measuring cefuroxime in bone. Cefuroxime penetration was impaired for all tissues, and bone may not be considered one distinct compartment.

Surface-modified functionalized polycaprolactone scaffolds for bone repair: in vitro and in vivo experiments.

A porcine calvaria defect study was carried out to investigate the bone repair potential of three-dimensional (3D)-printed poly-ε-caprolactone (PCL) scaffolds embedded with nanoporous PCL. A microscopic grid network was created by rapid prototyping making a 3D-fused deposition model (FDM-PCL). Afterward, the FDM-PCL scaffolds were infused with a mixture of PCL, water, and 1,4-dioxane and underwent a thermal-induced phase separation (TIPS) followed by lyophilization. The TIPS process lead to a nanoporous structure shielded by the printed microstructure (NSP-PCL). Sixteen Landrace pigs were divided into two groups with 8 and 12 weeks follow-up, respectively. A total of six nonpenetrating holes were drilled in the calvaria of each animal. The size of the cylindrical defects was h 10 mm and Ø 10 mm. The defects were distributed randomly using following groups: (a) NSP-PCL scaffold, (b) FDM-PCL scaffold, (c) autograft, (d) empty defect, (a1) NSP-PCL scaffold + autologous mononuclear cells, and (a2) NSP-PCL scaffold + bone morphogenetic protein 2. Bone volume to total volume was analyzed using microcomputed tomography (µCT) and histomorphometry. The µCT and histological data showed significantly less bone formation in the NSP-PCL scaffolds in all three variations after both 8 and 12 weeks compared to all other groups. The positive autograft control had significantly higher new bone formation compared to all other groups except the FDM-PCL when analyzed using histomorphometry. The NSP-PCL scaffolds were heavily infiltrated with foreign body giant cells suggesting an inflammatory response and perhaps active resorption of the scaffold material. The unmodified FDM-PCL scaffold showed good osteoconductivity and osseointegration after both 8 and 12 weeks.

Erythropoietin augments bone formation in a rabbit posterolateral spinal fusion model.

We tested the hypothesis that erythropoietin (EPO) enhances bone formation after posterolateral spinal fusion (PLF) in a rabbit model. Thirty-four adult rabbits underwent posterolateral intertransverse arthrodesis at the L5-L6 level using 2.0 g autograft per side. The animals were randomly divided into two groups receiving subcutaneous daily injections of either EPO or saline for 20 days. Treatment commenced 2 days preoperatively. Hemoglobin was monitored at baseline and 2, 4, and 6 weeks after fusion surgery. After euthanasia 6 weeks postoperatively, manual palpation, radiographic, and histomorphometric examinations were performed. Bone volume of the fusion mass was estimated by CT after 6 weeks. EPO increased bone fusion volume to 3.85 ccm (3.66-4.05) compared with 3.26 ccm (2.97-3.55) in the control group (p<0.01). EPO treatment improved vascularization of the fusion mass and increased hemoglobin levels (p<0.01). Fusion rate tended to be higher in the EPO group based on manual palpation, CT, and radiographic examinations. For the first time EPO has shown to augment bone formation after autograft PLF in a rabbit model. Increased vascularization provides a partial explanation for the efficacy of EPO as a bone autograft enhancer.

Estimating risk of pulmonary neoplastic embolism during vertebroplasty.

STUDY DESIGN: Vertebroplasty was simulated on a pig model. OBJECTIVE: To evaluate the risk of neoplastic tissue migration into lungs during vertebroplasty. SUMMARY OF BACKGROUND DATA: The application of vertebroplasty in spinal metastasis is not well documented. The risk of neoplastic tissue migration into the lungs during vertebroplasty remains unknown. METHODS: A cancer model was built in 11 Landrace pigs (50 kg) by injecting 99mTc-labeled albumin macroaggregates into the center of L5 and L6 prior to vertebroplasty. Continuous scintigraphic imaging was performed with 1-minute frames over the lungs and vertebrae before and after injection to ensure steady state and baseline. We surveyed free TcO4- in thyroid. Twenty minutes after the 99mTc injection, 2-level vertebroplasty was performed at L5 and L6 with 3 Jamshidi needles in each vertebra. Into each vertebra, on average, 2.8 ± 1.1 mL of poly(methyl methacrylate) cement (Depuy CMW, Blackpool, UK) was injected. Quantitative scintigrams were obtained within 90 minutes after vertebroplasty. X-rays and quantitative computed tomography scans quantified cement distribution. Means of 99mTc activity before and after vertebroplasty were compared in a paired t test. RESULTS: In this cancer model, we found an 80% risk of tissue migration to the lungs when performing vertebroplasty. In average, the study showed a significant amount of macroaggregate migration of 1.87% total range from 0% to 8% (CI: 0.05%-0.37%) with P = 0.045. There was no free TcO4- in the thyroid. Despite the standardized procedure, we found a large interindividual variation of pulmonary embolism. CONCLUSION: It is demonstrated that there exists a significant risk of exporting neoplastic disease or fatty tissue to the lungs when performing vertebroplasty. A similar adverse effect can be expected with balloon kyphoplasty. In patients with metastatic disease, vertebroplasty should be limited to those with short life expectancy.

Autologous stem cell therapy maintains vertebral blood flow and contrast diffusion through the endplate in experimental intervertebral disc degeneration.

STUDY DESIGN: Experimental, controlled, randomized, and paired study. OBJECTIVE: To evaluate regenerative effect of stem cell therapy on the vertebral endplate and introduce dynamic contrast-enhanced magnetic resonance imaging (MRI) as a tool in the investigation of endplate function. SUMMARY OF BACKGROUND DATA: The vertebral endplate plays a crucial role in nutritional supply to the intervertebral disc. Estimation of endplate function is an important parameter in future biologic therapy of intervertebral disc degeneration (IDD). METHODS: Four-level IDD was induced in each of 15 Gottingen minipigs. Percutaneous intradiscal injection of two hydrogels (Zimmer Biologics Inc, Austin, TX) and one loaded with stem cells was used as single interventions after 12 weeks. Total observation time was 24 weeks. MRI was performed before the initiation of treatment and killing of animals. RESULTS: Three animals were excluded because of spondylodiscitis. Stem cell and hydrogel treatment had significantly higher T2 values, relative vertebral blood flow and volume, as well as lower Pfirrmann scores when compared with degenerative controls. No statistical differences were found compared to normal controls. CONCLUSION: Stem cell and hydrogel therapy is able to partly regenerate IDD and maintain perfusion and permeability of the vertebral endplate and subchondral bone. Dynamic contrast-enhanced MRI may become an important tool in future investigation of the vertebral endplate.

Differences in early osteogenesis and bone micro-architecture in anterior lumbar interbody fusion with rhBMP-2, equine bone protein extract, and autograft.

PURPOSE: To investigate the microstructural differences and responsible mechanisms in early bone formation in anterior lumbar interbody fusion (ALIF) in the spine using rhBMP-2 (INFUSE), equine bone protein extract (COLLOSS E) or autograft. METHODS: Twelve Danish female landrace pigs underwent a 3-level ALIF procedure at L3-6. PEEK interbody cages packed with rhBMP-2, COLLOSS E, or autograft were inserted. The animals were divided into two groups of six, and observed for four and eight weeks postoperatively. MicroCT was performed for evaluation of microstructure of the bone within the cage. A mathematical finite element model was developed to investigate the aqueous behavior within the cages when exposed to external compressive forces. RESULTS: At 4 weeks postoperative bone surface volume fraction (BS/TV) using rhBMP-2 was higher than with use of COLLOSS E and autograft, while trabecular thickness (Tb.Th.) was lower using rhBMP-2 at this time-point. At eight weeks BS/TV and trabecular number (Tb.N.) were still higher using rhBMP-2 than autograft and COLLOSS E. Connectivity density was significantly higher using rhBMP-2 than using autograft or COLLOSS E at both time-points. Between four- and eight-week time-points BV/TV and Tb.Th. rose while Tb.N. declined using rhBMP-2. The degree of anisotropy and the calculated amount of trabeculae with main direction along the spinal axis, were higher at four weeks using COLLOSS E. rhBMP-2 had the highest amount of trabeculae directed along the spinal axis at eight weeks. A change in main direction between four and eight weeks was observed for both autograft and rhBMP-2. The numerical results from the finite element model verify that significantly different flow pattern emerges as the boundary conditions are altered. At four weeks there was an evident correlation between trabecular orientation and flow pattern using rhBMP-2. CONCLUSION: This study reveals large differences in microstructure in the early osteogenesis and explains important mechanisms of early bone formation using rhBMP-2, COLLOSS E or autograft treatment. These differences might explain some of the unfortunate events reported such as edema, swelling, and excessive bone formation using different bone graft substitutes in spinal fusion procedures.

Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion.

To elucidate the molecular mechanisms of spinal fusion with different graft materials during an anterior lumbar interbody fusion, we examined the gene-expression profiles after implantation of equine bone protein extract, rhBMP-2 and autograft using microarray technology and data analysis, including hierarchical clustering, self-organizing maps (SOM), KEGG pathway and Biological process GO analyses in a porcine model. The results suggest that equine bone protein extract exhibited a more similar expression pattern with autograft than that of rhBMP-2. rhBMP-2 recruits progenitor cells, proliferation and differentiation possibly by inducing various factors including PGHS-2, IFGBP-2, VEGF and chemokines and then leads to preferable membranous ossification and bone remodeling. Conversely, equine bone protein extract results in endochondral ossification via upregulation of cartilage-related genes. Ossification by inducing direct osteoblastic differentiation and obviating the cartilaginous intermediate phases may increase spinal fusion rate.

ISSLS prize winner: positron emission tomography and magnetic resonance imaging for monitoring interbody fusion with equine bone protein extract, recombinant human bone morphogenetic protein-2, and autograft.

STUDY DESIGN: Prospective and randomized experimental study with anterior lumbar interbody fusion in a porcine model. OBJECTIVE: To assess the early time-course of spinal fusion with equine bone protein extract (COLLOSS E), recombinant human bone morphogenetic protein-2 (rhBMP-2), and autograft using quantitative methods of positron emission tomography (PET)/computed tomography and magnetic resonance imaging (MRI). SUMMARY OF BACKGROUND DATA: Different growth and differentiation factors are currently being used for inducing bone formation in spinal fusion. However, the mechanisms and time-course of bone formation using these graft substitutes remain less known. METHODS: Eighteen female Danish landrace pigs underwent a 3-level anterior lumbar interbody fusion procedure from L3-L6. A PEEK cage, packed with COLLOSS E, rhBMP-2, or autograft, was randomly placed. Each group of 6 pigs was observed for 2, 4, or 8 weeks, respectively. F PET/computed tomography and MRI examinations were performed, and data were correlated with histomorphometry. PET data were analyzed using a Gjedde-Patlak plot. K-values from the plot correspond to the metabolic rate. T2-values were calculated by T2 mapping. RESULTS: rhBMP-2 presented the highest bone formation on histologic sections at 25.6% at 4 weeks after surgery. Eight weeks after surgery, autograft had the highest bone formation with 37.3%, which was significantly higher than rhBMP-2 at 30.5% (P < 0.05), and higher than COLLOSS E at 27.0% (P = 0.06). COLLOSS E and rhBMP-2 had significantly higher K-values than autograft (P < 0.05) at 2 weeks after surgery. There were no differences in K-values between COLLOSS E and autograft at 4 and 8 weeks. However, rhBMP-2 was significantly higher at 4 weeks and lower at 8 weeks than these 2 (P < 0.05). Linear correlation, R = 0.8275, was observed for intertrabecular volume/total volume and T2-values. CONCLUSION: PET and MRI are valid tools for monitoring the process of interbody fusion in vivo. Osteogenic mechanisms using COLLOSS E resembles that of autograft by the process of endochondral ossification. rhBMP-2 deposits osteoid directly on the collagen network.