Bone tissue engineering constructs (BTEC) combining natural resorbable osteoconductive scaffolds and mesenchymal stem cells (MSCs) have given promising results to repair critical size bone defect. Yet, results remain inconsistent. Adjonction of an osteoinductive factor to these BTEC, such as rh-BMP-2, to improve bone healing, seems to be a relevant strategy. However, currently supraphysiological dose of this protein are used and can lead to adverse effects such as inflammation, ectopic bone and/or bone cyst formation. Interestingly, in a preliminary study conducted in ectopic site in a murine model, a synergistic effect on bone formation was observed only when a low dose of rh-BMP-2 was associated with MSCs-seeded coral scaffolds but not with a high dose. The objective of the study was then to evaluate a BTEC combining coral scaffold, MSCs and a low dose of rh-BMP-2 in a large animal model of clinical relevance. Sixteen sheep were used for this study. MSCs were isolated from an aspirate of bone marrow harvested from the iliac crest of each sheep receiving BTEC with MSCs, cultivated and seeded on Acroporacoral scaffolds one week before implantation. Rh-BMP-2, used at two different doses (low dose: 68μg/defect and high dose: 680μg/defect), was diluted and absorbed on Acroporacoral scaffold one day before implantation. Metatarsal segmental bone defects (25 mm) were made in the left metatarsal bone of the sheep, stabilized by plate fixation, and filled with Acroporacoral scaffolds loaded with either (i) MSCs and a low dose of rh-BMP-2 (Group 1;n=6), (ii) a low dose of rh-BMP-2 (Group 2;n=5), (iii) a high dose of rh-BMP-2 (Group 3;n=5). Standard radiographs were taken after each surgery and each month until sheep sacrifice, 4 months postoperatively. Bone healing and scaffold resorption were assessed by micro-computed-tomography (μCT) and histomorphometry. Results were compared to a historical control group in which coral scaffolds were loaded with MSCs. Bone volumes (BV) evaluated by μCT and bone surfaces (BS) evaluated by histomorphometry did not differ between groups (BV: 1914±870, 1737±841, 1894±1028 and 1835±1342 mm. 3. ; BS: 25,41±14,25, 19,85±8,31, 25,54±16,98 and 26,08±22,52 %; groups 1, 2, 3 and control respectively); however, an higher bone union was observed in group 1 compared to the others (3, 1, 2 and 2 sheep with bone union in groups 1, 2, 3 and control respectively). No histological abnormalities were observed in any group. Coral resorption was almost complete in all specimens. No significant difference in coral volumes and coral surfaces was observed between groups. A trend towards a higher variability in coral resorption was noted in group 1 compared to the others. There seems to be a benefit to associate low dose of rh-BMP-2 with MSCs-seeded coral scaffolds as this strategy allowed an increase of bone unions in our model. Yet, results remain inconsistent. Although, defective coupling between scaffold resorption and bone formation impaired bone healing in some animals, adjunction of rh-BMP-2 (even at low dose) to CSMs loaded construct is a promising strategy for bone tissue engineering

We have evaluated the effect of the short-term administration of low therapeutic doses of modern COX-2 inhibitors on the healing of fractures. A total of 40 adult male New Zealand rabbits were divided into five groups. A mid-diaphyseal osteotomy of the right ulna was performed and either normal saline, prednisolone, indometacin, meloxicam or rofecoxib was administered for five days. Radiological, biomechanical and histomorphometric evaluation was performed at six weeks. In the group in which the highly selective anti-COX-2 agent, rofecoxib, was used the incidence of radiologically-incomplete union was similar to that in the control group. All the biomechanical parameters were statistically significantly lower in both the prednisolone and indometacin (p = 0.01) and in the meloxicam (p = 0.04) groups compared with the control group. Only the fracture load values were found to be statistically significantly lower (p = 0.05) in the rofecoxib group. Histomorphometric parameters were adversely affected in all groups with the specimens of the rofecoxib group showing the least negative effect. Our findings indicated that the short-term administration of low therapeutic doses of a highly selective COX-2 inhibitor had a minor negative effect on bone healing

Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats. Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT. Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling. The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects

Background. Bone Morphogenetic Protein (BMP) has been used in clinical practice to stimulate fracture healing and spinal arthrodesis. Difficulty in localising and maintaining BMP at the target site has resulted in the use of large doses of BMP, and has been associated with significant adverse effects. We have previously shown clay hydrogels can bind growth factors for localised efficacy. We hypothesised that localisation of BMP within clay gels would reduce the dose required to mediate bone formation. Methods. 2×10-4mg and 1×10-5 mg BMP were mixed in Laponite and applied to collagen sponge. 3 sponges containing high dose, and 3 containing low dose BMP were implanted subcutaneously in a mouse. This process was repeated in 8 mice, for controls, alginate hydrogel was used in a further 8 mice, and 1 mouse received 6 blank collagen scaffolds. Micro Computed Tomography was used to assess bone formation fortnightly; at 8 weeks the mice were culled and underwent histological analysis. Results. Mean Bone Volume formed within collagen per μg BMP was significantly greater with Laponite and low dose BMP compared to Alginate and Laponite with high dose BMP (p<0.0001). No bone formation was observed with Alginate and low dose BMP. Conclusions. We have demonstrated that Laponite is able to reduce, by several orders, the effective dose of BMP required to mediate ectopic bone formation compared to current gold standard methods of BMP delivery. Clinical translation of this finding offers, potentially, great significance to orthopaedic surgery. Level of Evidence. In vivo study. Approval. Our study received ethical approval complied with Home Office licensing. Acknowledgments. Funded by grants from EU(FP7) Biodesign, Rosetrees Trust, BBSRC and EPSRC

Objectives. This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols. Methods. Two manual preparation procedures (single-spin (SS) at 900 g for five minutes; double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects. Both preparations were tested for platelet activation by one of three activation protocols: no activation, activation with calcium (Ca) only, or calcium with a low dose (50 IU per 1 ml PRP) of thrombin. Each preparation was divided into four aliquots and incubated for one hour, 24 hours, 72 hours, and seven days. The cytokine-release kinetics were evaluated by assessing PDGF, TGF, VEGF, FGF, IL-1, and MMP-9 concentrations with bead-based sandwich immunoassay. Results. The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols. Ca-only activation had a significant effect on the DS PRPs (where the VEGF, FGF, and IL-1 concentrations were sustained) while Ca/thrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short). The IL-1 content showed a significant increase with Ca-only or Ca/thrombin activation while these activations did not increase the MMP-9 concentration. Conclusion. The SS and DS methods differed in their effect on cytokine release, and this effect varied among the cytokines analysed. In addition, low dose of thrombin/calcium activation increased the overall cytokine release of the PRP preparations over seven days, relative to that with a calcium-only supplement or non-activation. Cite this article: Professor J. H. Oh. Cytokine-release kinetics of platelet-rich plasma according to various activation protocols. Bone Joint Res 2016;5:37–45. doi: 10.1302/2046-3758.52.2000540

Abstract. Objectives. Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype. Results. Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNAseq revealed that there were 2,085 highly significant upregulated genes in 3D matrices compared to TCP. Notably, >90% of highly expressed genes (including FOSB, FOS and TNFAIP6) were shared in all hydrogels. Gene ontology confirmed the TNF signalling pathway among the most significantly represented. Protein-protein interaction predictions identified TNF-alpha/NF-kappa B and AP1 pathways as differentially influenced by the hydrogel environment. Using inhibitors to these pathways, NFkB, but not AP1, impacted on the upregulation of TNFAIP6 and CXCL10 in 3D culture. Conditioned media from these studies was added to cultures of human monocytes with distinct changes in the resulting macrophage phenotype. MSCs in a 3D environment promoted a greater acquisition of the M2 repair macrophage phenotype and impacted on the numbers of pro-inflammatory M1 macrophages. Conclusion. These data provide further evidence that the immunomodulatory action of human MSCs can be influenced by the surrounding structural environment. These observations have significance for understanding the events that following skeletal injury and the potential to be exploited in preconditioning MSCs for cell therapy

Summary Statement. This study explores the therapeutic use of MSCs to enhance ligament healing from an immuno-modulatory perspective. We report improved healing with MSC treatment, but inconsistent effects on inflammatory markers. Introduction. Mesenchymal stem cell (MSC) use continues to hold untapped potential as a therapeutic agent because: 1) MSCs have the ability to differentiate into several different connective tissues such as cartilage, bone, muscle and fat (1–3), and 2) MSCs can modulate immune and inflammatory responses that affect healing (4, 5). This paradigm shift from differentiation to immune modulation is being studied for different applications (6). Several studies suggest MSCs decrease inflammation by reducing pro-inflammatory cytokines and changing the macrophage phenotype from M1 (classically-activated) to M2 (alternatively-activated) (7–10). However, their immune-modulatory effects within a healing ligament remain unexplored. MSCs can behave differently depending on the tissue and healing environment they encounter, which leads to our interest in MSC immune-modulation in healing ligaments. Methods. Forty-four rats underwent bilateral MCL transection. Days 5 and 14 healing were examined comparing two cell doses (1×10. 6. MSCs or 4×10. 6. MSCs). At the time of surgery, fluorescently-labeled rat MSCs (passage 8–10) were injected into the right MCL, while the left MCL served as a control for normal healing. MCLs were collected at the different time points and processed with immunohistochemistry (n=12). Type 1 macrophages (M1) and type 2 macrophages (M2) were quantified spatially within the healing ligaments. Twelve rats with MSC injections underwent mechanical testing. A multiplex cytokine reader measured 10 different cytokines in the healing ligaments at days 5 and 14. Results. MSCs were detected solely in the healing region and healing region edges at Days 5 and 14 in both dose groups using fluorescence microscopy. At day 5, the higher dose of cells produced significant M2 changes throughout the ligament. There were more M2′s (p=.05) in the distal and proximal healing regions of the normal healing ligament compared to the MSC injection group. There were significant changes in both the low dose and high dose groups at day 14. Fewer M1′s were found in the ends (p=.01) and throughout the MCL (p=.04) in the low dose group. M2′s were decreased in the ends (p=.04), but only in the ligaments that received the higher dose of MSCs. Cytokine analysis showed a greater amount of pro-inflammatory cytokines in the high dose MSC group at Day 5 (IL-1β, IL-2, and Interferon-Y) compared to controls, along with increased IL-12 at Day 14. The low dose MSC injection group demonstrated increased strength with an average failure load of 26.4N compared to 20.9N in the control group (p=.03). Low dose ligaments also exhibited increased stiffness with an average of 12.2 N/mm compared to 10.0 N/mm (p=.01) in control ligaments. Discussion. MSCs improved healing when applied at an appropriate dose as shown by improved mechanical properties at day 14. Interestingly, the smaller dose of 1 million cells proved more successful than the larger dose of 4 million cells. MSCs also affected the cytokine profile and macrophage phenotype at both healing time points, but not always as expected with regard to inflammatory cells and cytokines

Background. Stem cell based intervertebral disc (IVD) regeneration is quickly moving towards clinical applications. However, many aspects need to be investigated to routinely translate this therapy to clinical applications, in particular, the most efficient way to deliver cell to the IVD. Cells are commonly delivered to the IVD through the annulus fibrosus (AF) injection. However, recent studies have shown serious drawbacks of this approach. As an alternative we have described and tested a new surgical approach to the IVD via the endplate-pedicles (transpedicular approach). The Purpose of the study was to test MSCs/hydrogel transplantation for IVD regeneration in a grade IV preclinical model of IDD on large size animals via the transpeducular approach with cell dose escalation. Methods. Adult sheep (n=18) underwent bone marrow aspiration for autologous MSC isolation and expansion. MSC were suspended in autologous PRP and conjugated with Hyaluronic Acid and Batroxobin at the time of transplant (MSCs/hydrogel). Nucleotomy was performed via the transpedicular approach in four lumbar IVDs and that were injected with 1) hydrogel, 2) Low doses of MSC/hydrogel, 3) High doses of MSC/hydrogel, 4) no injection (CTRL). The endplate tunnel was sealed using a polyurethane scaffold. X-ray and MRI were performed at baseline and 1,3,6,12 months. Disc macro- and micro-morphology were analysed at each time point. Results. The MRI index showed a significant decrease in the untreated group, the disc injected with hydrogel and those injected with low MSC dose compared to healthy discs in all time points. The discs treated with high dose of MSC showed maintenance of the MRI index compared to the healthy disc. Morphologically, the grade of degeneration evaluated using the were in agreement with the grades observed at the MRI. Conclusions. An effective dose of autologous MSC (1−107 cell/ml) delivered via the alternative transpedicular approach regenerates the NP in a preclinical model of grade IV IDD maintaining the AF intact This preclinical study has high translational value as large animal model with the long fallow up were used, MSCs were expanded in GMP facility simulating the clinical scenario, and the hydrogel were composed of clinically available drags and materials

Long bone fractures in patients with diabetes mellitus (DM) are slow to heal, often resulting in delayed reunion or non-union. It is reasonable to postulate that the underlying cause of these DM-associated complications is a reduced population of bone marrow progenitor cells and/or their dysfunction. With the hypothesis that the administration of healthy, allogeneic adult bone marrow-derived mesenchymal stromal cells (MSCs) can enhance DM fracture healing, the aim of this endeavour was to assess the efficacy of MSC administration to support fracture repair using two doses. Here 250,000 or 500,000 human bone marrow-derived MSCs were locally introduced to femoral fractures in diabetic mice, and the quality of de novo bone assessed 8 weeks later. Preliminary bone bridging was evident in all animals; however, a large circumferential reparative callus was consistently retained indicating non-union. Micro-CT analysis elucidated consistent callus dimensions, bone mineral density, bone volume/total volume in all groups, but an increase in bone surface area/bone volume in cell-treated fractures. Moreover, greater amounts of mature bone were identified in fractures treated with a low dose of MSCs. Four-point bending evaluation of the mechanical integrity of the repairing fracture indicated a statistically significant improvement in flexure strength and flexure modulus in DM fractures treated with 250,000 MSCs as compared to controls. An improvement in total energy required for failure was observed in both groups that received MSCs. Therefore, the administration of non-DM bone marrow-derived MSCs supported the development of more mature bone in the reparative callus, resulting in greater mechanical integrity

Metaphyseal fracture healing is important in joint-adjacent fractures and appears to differ from diaphyseal healing. We recently found that a biomaterial delivering bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA) healed the metaphyseal bone in a tibial defect but failed closing the cortical defect. In this study we added a BMP-2 soaked collagen membrane to study cortical healing from the muscle tissue surrounding the bone. We used SD rats and a 4.5 mm metaphyseal circular tibial defect. In group 1 (G1), a porous gelatin-calcium sulphate-hydroxyapatite (GCH) biomaterial containing rhBMP-2 and ZA was used to fill the defect (GCH+5 μg BMP-2+10 μg ZA). In group 2 (G2), we used a collagen membrane (2 μg BMP-2) to cover the GCH filled defect (GCH+3μg BMP+10 μg ZA). Group 3 (G3) was an empty control. Animals were sacrificed after 8-weeks and bone regeneration was evaluated with micro-CT and histology. In both G1 (P<0.001) and G2 (p<0.001) a significantly higher mineralized volume was found in the defect compared to empty G3. In G2 higher mineralized volume was found in the cortical region compared to both G1 (p<0.01) and G3 (p<0.001) as seen via micro-CT. Histologically, G1 and G2 showed islands of trabecular bone in the defect peripherally but only G2 showed cortical healing. G3 was empty in the middle but showed healed cortex. In conclusion, GCH can be used to deliver BMP-2 and ZA to promote metaphyseal bone growth. A membrane (CM) doped with low dose BMP-2 improved cortical regeneration

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect. Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1

Polyether ether ketone (PEEK) has been increasingly employed as biomaterials for trauma, orthopeadic, and spinal implants. However, concern has been raised about the inertness of PEEK which limits bone integration. In this study, we have coated PEEK with a functional material seeking to promote osteogenic differentiation of human mesenchymal stem cells (hMSC). We have used spray drying to coat poly(ethyl acrylate) (PEA) as a coating on PEEK. This technique is simple, allows a range of controlled coating thicknesses (from hundred nm to a few um), cost effective and easily translatable to scaffolds or implant surfaces for existing or new orthopaedic applications. PEA induces the organisation of fibronectin (FN) into nanonetworks upon simple adsorption from protein solutions. These FN nanonetworks on PEA represent a microenvironment for efficient growth factor binding and presentation in very low but effective doses. In this study we show cell adhesion and stem cell differentiation towards an osteogenic lineages when bone morphogenetic protein 2 (BMP2) was adsorbed on these engineered PEEK/PEA/FN microenvironments in very low doses. Overall, the developed functional coatings on PEEK has the potential to allow the translation of this material into orthopaedic applications

Background. The doses of local rhBMP-2 in commercially available materials are high with known drawbacks such as inflammation and premature bone resorption. The latter can be prevented by adding bisphosphonates like zoledronic acid (ZA) but systemic ZA has side effects and patient adherence to treatment is low. In a recent study, we have shown that local co-delivery of rhBMP-2 and ZA via a calcium sulphate/hydroxyapatite (CS-HA) biomaterial can be used to regenerate both cortical and trabecular bone in a rat model of metaphyseal bone defect. Even low doses of local ZA in the biomaterial showed promising results and increased bone formation within the defect compared to the controls. A step before clinical translation of the local treatment regimen is to evaluate the in-vivo release kinetics of these additives and thus in this study, we aimed to investigate the in-vivo pharmacokinetics of rhBMP-2 and ZA from the CS-HA biomaterial in a rat abdominal muscle pouch model over a period of 4-weeks. Methods. In-vivo release kinetics of 125I labeled rhBMP-2 and 14C labeled ZA was performed using an abdominal muscle pouch model in rats (n=6). Both rhBMP-2 and ZA were labeled commercially with a radiochemical purity of >95%. The detection of 125I -rhBMP-2 release was performed by implanting pellets of the CS-HA biomaterial containing 125I -rhBMP-2 and ZA and the same animals followed over a period of 4-weeks (day 1, 3, 7, 14, 21& 28) using SPECT imaging. Similarly, the 14C-ZA was detected by implanting CS-HA pellets containing rhBMP-2 and 14C-ZA. Release was detected via scintillation counting and at each time point (Day 1, 7, 14& 28) 6-animals were sacrificed. Results. BMP Release. The CS-HA biomaterial retained 95±11% after 3-days, 88±12% after a week, 66±9% after 2 weeks, 51±5% after 3 weeks and 43±7% of 125I labeled rhBMP-2 after 4-weeks in-vivo (SPECT-CT). ZA Release. The CS-HA biomaterial retained 89±14% after a week, 84±8% after 2 weeks, 83±9% after 3 weeks and 77±3% of 14C labeled ZA after 4 weeks of in-vivo implantation. Discussion. Improved carriers and better knowledge of the release might improve the effect of bone active drugs in orthopedics. Our previous study shows that an off-the-shelf ceramic biomaterial combined with ZA alone or with both rhBMP-2 and ZA can be used to regenerate bone with potential for clinical translational. This study demonstrates long-term co-delivery of both rhBMP-2 and ZA in-vivo via the biomaterial. Constant availability of rhBMP-2 over a long period of time can give osteoinductive properties to the material while presence of local ZA prevents premature bone loss. The pharmacokinetic release pattern differs from what we have reported in vitro with less BMP and more ZA being released in vivo during the first 4 weeks. We speculate that rapid protein passivation of the ceramic material slows the release of BMP and partly preventing the ZA binding to apatite

Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals. We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs

The function of the knee joint is to allow for locomotion and is comprised of various bodily structures including the four major ligaments; medial collateral ligament (MCL), lateral collateral ligament (LCL), anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL). The primary function of the ligaments are to provide stability to the joint. The knee is prone to injury as a result of osteoarthritis as well as ligamentous and meniscal lesions. Furthermore, compromised joint integrity due to ligamentous injury may be a result of direct and indirect trauma, illness, occupational hazard as well as lifestyle. A device capable of non-invasively determining the condition of the ligaments in the knee joint would be a useful tool to assist the clinician in making a more informed diagnosis and prognosis of the injury. Furthermore, the device would potentially reduce the probability of a misdiagnosis, timely diagnosis and avoidable surgeries. The existing Laxmeter prototype (UK IPN: GB2520046) is a Stress Radiography Device currently limited to measuring the laxity of the MCL and LCL at multiple fixed degrees of knee flexion. Laxity refers to the measure of a ligament's elasticity and stiffness i.e. the condition of the ligament, by applying a known load (200N) to various aspects of the proximal tibial and thereby inducing tibial translation. The extent of translation would indicate the condition of the ligament. The Laxmeter does not feature a load applying component as of yet, however, it allows for the patient to be in the most comfortable and ideal position during radiographic laxity measurement testing. The entire structure is radiolucent and attempts to address the limitations of existing laxity measurement devices, which includes: excessive radiation exposure to the radiographic assistant, little consideration for patient ergonomics and restrictions to cruciate or collateral ligament laxity measurements. The study focusses on further developing and modifying the Laxmeter to allow for: the laxity measurement of all four major ligaments of the knee joint, foldability for improved storage and increased structural integrity. Additionally, a load applicator has been designed as an add-on to the system thereby making the Laxmeter a complete Stress Radiography Device. Various materials including Nylon, Polycarbonate, Ultra High Molecular Weight Polyethylene (UHMWPE) – PE 1000, and Acetal/ POM were tested, using the Low Dose X-ray (Lodox) scanner, to determine their radiolucency. All materials were found to be radiolucent enough for the manufacture of the Laxmeter structure as well as the load applicator in order to identify and measure the translation of the tibia with respect to the stationary femur. The Laxmeter allows for the measurement of the laxity of the MCL and LCL at multiple fixed degrees of flexion by providing the ideal patient position for testing. The next iteration of the device will present an affordable and complete Stress Radiography Device capable of measuring the laxity of all four major ligaments of the knee joint at multiple fixed degrees of flexion. Future work would include aesthetic considerations as well as an investigation into carbon-fibre-reinforced plastics

Currently, the cement being used for hemiarthroplasties and total hip replacements by the authors and many other surgeons in the UK is Palacos® (containing 0.5g Gentamicin). Similar cement, Copal® (containing 1g Gentamicin and 1g Clindamycin) has been used in revision arthroplasties, and has shown to be better at inhibiting bacterial growth and biofilm formation. We aim to investigate the effect on SSI rates of doubling the gentamicin dose and adding a second antibiotic (clindamycin) to the bone cement in hip hemiarthroplasty. We randomised 848 consecutive patients undergoing cemented hip hemiarthroplasty for fractured NOF at one NHS trust (two sites) into two groups: Group I, 464 patients, received standard cement (Palacos®) and Group II, 384 patients, received high dose, double antibiotic-impregnated cement (Copal®). We calculated the SSI rate for each group at 30 days post-surgery. The patients, reviewers and statistician were blinded as to treatment group. The demographics and co-morbid conditions (known to increase risk of infection) were statistically similar between the groups. The combined superficial and deep SSI rates were 5 % (20/394) and 1.7% (6/344) for groups I and II respectively (p=0.01). Group I had a deep infection rate 3.3 %(13/394) compared to 1.16% (4/344) in group II (p=0.082). Group I had a superficial infection rate 1.7 % (7/394) compared to 0.58% (2/344) in group II (p=0.1861). 33(4%) patients were lost to follow up, and 77 (9%) patients were deceased at the 30 day end point. There was no statistical difference in the 30 day mortality, C. difficile infection, or the renal failure rates between the two groups. Using high dose double antibiotic-impregnated cement rather than standard low dose antibiotic-impregnated cement significantly reduced the SSI rate (1.7% vs 5%; p=0.01) after hip hemiarthroplasty for fractured neck of femur in this prospective randomised controlled trial

Summary Statement. Antioxidant containing UHMWPE particles induced similar levels of in vitro macrophage proliferation and in vivo inflammation in the mouse air pouch model as UHMWPE particles alone. Benefit of antioxidant in reducing wear particle induced inflammation requires further investigation. Introduction. Wear particles derived from UHMWPE implants can provoke inflammatory reaction and cause osteolysis in the bone, leading to aseptic implant loosening. Antioxidants have been incorporated into UHMWPE implants to improve their long term oxidative stability. However it is unclear if the anti-inflammatory property of the antioxidant could reduce UHMWPE particle induced inflammation. This study evaluated the effect of cyanidin and vitamin E on UHMWPE induced macrophage activation and mouse air pouch inflammation. Methods. Four types of UHMWPE were used: (1) compression molded (CM) conventional GUR1020 (PE); (2) CM GUR1020 blended with 300 ppm cyanidin (C-PE); (3) CM GUR1020 blended with 1000 ppm α-tocopherol (BE-PE); and (4) CM GUR1020, gamma irradiated at 100kGy, diffused with α-tocopherol, and sterilised at 30kGy (DE-PE). Particles were generated by cryomilling. Particle count, size, and aspect ratio were determined using SEM and Image Pro. Each particle group was cultured with RAW264.7 macrophage cells at four concentrations (0.625, 1.25, 2.5, and 5 μg/mL) in a standard medium for 4 days. Cell numbers were quantified using MTT assay. Cytokine expression (IL-1β, TNFα, and IL-6) was measured using RT-PCR and ELISA. Particles were also suspended in PBS at 2 concentrations (0.2 or 1 mg) and injected into subcutaneous air pouches in BALB/c mice. Control animals were injected with PBS alone. Six days post-injection air pouches were harvested, half of which were fixed for histology to measure membrane thickness and inflammatory cell quantity. Remaining air pouches were frozen and analyzed by ELISA for cytokine production. Data were analyzed using one-way ANOVA with post hoc testing. P<0.05 was considered significant. Results. All 4 materials showed similar particle characteristics after cryomilling. Particle size ranged from 1 to 19 μm with 33% of particle population smaller than 2 μm. All particle groups supported macrophage proliferation, showing an inverse correlation between proliferation rate and particle dose. Gene expression of IL-1β and TNFα also showed an inverse correlation with particle dose. Expression of IL-1β, TNFα, and IL-6 appeared lower in cells cultured with C-PE than the other 3 materials. The accumulative protein productions of IL-1β and TNFα were significantly lower while IL-6 production was moderately lower in C-PE, BE-PE and DE-PE when compared to PE. Injection of polyethylene particles increased the air pouch membrane thickness significantly compared to the PBS control in all particle types and doses. Higher particle dose induced thicker membrane in all 4 materials. A similar trend was also observed in the percentage of inflammatory cell infiltration in the pouch membrane. C-PE and DE-PE particles at low dose and C-PE particles at high dose induced lower levels of IL-1β and TNFα than PE. IL-6 production was similar between PE and other 3 groups. Discussion/Conclusion. Antioxidant incorporated in UHMWPE did not alter the level of macrophage proliferation and air pouch inflammation induced by UHMWPE particles, although it reduced cytokine gene expression. Future investigation in a synovial joint environment is desired to evaluate the chronic inflammation response to antioxidant containing UHMWPE wear particles and to verify the effect of antioxidant in UHMWPE properties

Summary Statement. The current study introduced the effects of projection errors on ankle morphological measurements using CT-based simulated radiographs by correlation analysis between 2D/3D dimensions and reliability analysis with randomised perturbations while measuring planar parameters on radiographs. Introduction. Clinical success of total ankle arthroplasty (TAA) depends heavily on the available anatomy-based information of the morphology for using implants of precisely matched sizes. Among the clinically available medical imaging modalities, bi-planar projective radiographs are commonly used for this purpose owing to their convenience, low cost, and low radiation dose compared with other modalities such as MRI or CT. However, the intrinsic articular surface of the ankle joint is not symmetrical and oblique which implies that it is difficult to describe all the anatomical dimensions in detail with only one radiograph, thereby hindering the determination of accurate ankle morphometric parameters. The purposes of this study were to compare the measurements of ankle morphology using 3D CT images with those on planar 2D images; and to quantify the repeatability of the 2D measurements under simulated random perturbations. Patients & Methods. Fifty-eight fresh frozen cadaveric ankle specimens were used in the current study. Each specimen was fixed in the neutral position with a plastic frame. After fixation, the specimen-fixation construct was scanned using a 16-slice spiral CT scanner (GE BrightSpeed 16, C&G Technologies, USA) with a slice thickness of 0.625 mm. A global coordinate system was embedded in the ankle specimen with the origin at the geometric center of the talus, the anteroposterior (A/P) axis in parallel to the base-plate, the superoinferior (S/I) axis perpendicular to the base-plate, and the mediolateral (M/L) axis as the line perpendicular to both the A/P and S/I axes. Fourteen 3D morphological parameters were automatically determined using a house-developed program in MATLAB R2010a (The MathWorks, Inc., USA). A simulated standard digital radiography system, in which the X-ray focus was 1 meter away from the image plane, was also introduced to determine the planar 2D morphological parameters for comparing with those determined in 3D. Reliability with randomised perturbations during measurements was also assessed in terms of the intra-class correlation coefficients using a 2-way mixed-effects average model (ICC3, k) for intra-examiner assessments. All statistical analysis was performed using SPSS 13.0 (SPSS Inc., USA). Results. Most of the morphological parameters had high correlation and reliability, except for the maximal tibial thickness (MTiTh), distance between most vertex of tibial mortise to the level of MTiTh (MDV) and radius of trochlea tali (TaR) had moderate to low correlation which were 0.54, 0.37 and 0.09 respectively. The ICC coefficients indicated that the MDV, talus width (TaW) and inclination angle between two most vertex points of trochlea tali (MLATa) had moderate and poor reliability which were 0.59, 0.49 and 0.07 respectively. Discussion/Conclusion. The current study introduced the effects of projection errors on ankle morphological measurements using CT-based simulated radiographs by correlation analysis between 2D/3D dimensions and reliability analysis with randomised perturbations while measuring planar parameters on radiographs. MTiTh and MDV are the important parameters to help surgeon pre-surgical decision-making. TaW is one of the critical parameters for choosing accurate sise of TAA implant. It implies that the respectively accurate pose of ankle is critical during bi-planar radiography

Objectives

The aim of this study was to investigate the effect of granulocyte-colony stimulating factor (G-CSF) on mesenchymal stem cell (MSC) proliferation in vitro and to determine whether pre-microfracture systemic administration of G-CSF (a bone marrow stimulant) could improve the quality of repaired tissue of a full-thickness cartilage defect in a rabbit model.

Objectives

Recent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis.