Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (ClinicalTrials.gov Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (ClinicalTrials.gov Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress.

Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540.

Bone has a remarkable capacity to heal. However, in some instances the amount of bone which is needed to heal exceeds its healing capacity. Due to reported issues with current treatments there is continued research into alternative approaches with a view to producing an off the shelf alternative to the gold standard autologous bone transplants. The current investigated the use of a chitosan/hydroxyapatite scaffold, which was used to covalently bone morphogenetic protein and vascular endothelial growth factor using a UV crosslinking process. Results indicate that the incorporation of hydroxyapatite increased the mechanical properties of the scaffold compared to chitosan alone. Furthermore, crosslinking was confirmed using swelling studies and FTIR analysis. Elisa indicated that physiological doses of BMP were released after 10 days while in vitro testing did not indicate a cytotoxic response to the scaffold. In vivo testing in a rat femoral defect model indicated the efficacy of the treatment with scaffolds containing BMP and VEGF in combination resulting in more bone in the defect compared to the scaffold alone 8 weeks post-surgery.

NICE guidelines state that patients undergoing hip or knee arthroplasty should start as an in-patient and then continue pharmacological VTE prophylaxis for 28–35 days.

Retrospective review of all elective hip and knee arthroplasties during one calendar month gave a baseline measurement of how many patients had VTE prophylaxis prescribed on their discharge summary.

A new, electronically completed, bespoke Trauma and Orthopaedic discharge summary was created with a discreet area clearly marked for VTE prophylaxis, to serve as a reminder to prescribe it.

In March 2012, 93 patients underwent hip/knee arthroplasty. 76% (71/93) were prescribed VTE prophylaxis to take home, there was no clinical reason explaining the failure to prescribe prophylaxis in the remaining 24%.

In July 2013, after implementation of the change, 117 patients underwent hip/knee arthroplasty. 99% (116/117) were prescribed VTE prophylaxis to take home.

Repeat audit in October 2013 showed that 103 patients underwent hip/knee arthroplasty and 100% were prescribed VTE prophylaxis.

A simple but clear change to paperwork, brought about a rapid and seemingly lasting change in the prescription of out-patient VTE prophylaxis.

The improvement was seen before and after a change of the Junior Doctor workforce suggesting the change in documentation was the main influencing factor.

Introduction

Review the results of modified Lautenbach procedure (new method) to treat chronic osteomyelitis of the long bones.

Background: High doses of local antibiotics are used to treat infected acute fractures or chronic osteomyelitis. In the U.S.A., tobramycin is one of the most commonly used antibiotics in trauma surgery. It is an aminoglycoside antibiotic with a broad spectrum of action. However, its effect on the osteogenic potential of bone marrow derived mesenchymal stem cells (MSC’s) is unknown. We hypothesised that high concentrations of tobramycin would be detrimental to the osteogenic potential of multipotent stem cells derived from the bone marrow.

Methods: MSC’s were derived in vitro from reamings obtained in patients undergoing hip hemiarthroplasties. Following subculture, these cells were exposed to various concentrations of tobramycin for 15 days, with a change of media every other day.

The amount of bone formed under each condition was assessed by solubilising the mineral content in hydrochloric acid overnight and then measuring the change in colour induced by Calcium exposed to a commercial reagent. The amount of calcium detected was then determined using a standard curve.

This experiment was repeated in cells from 3 patients.

Results: The amount of calcium formed was as follows Tobramycin concentration of 0 microg/ml

There was a statistically significant impairment in osteogenesis at a concentration of tobramycin of 400 microg/ml and above.

Conclusion: A high local dose of tobramycin affects negatively the osteogenic potential of stem cells derived from the bone marrow.

The Ilizarov frame is a circular external fixator, invented by Professor Ilizarov in Siberia during the 1950’s. It uses the principle of distraction osteogenesis to form new bone in a variety of clinical situations where bone lengthening or realignment is needed. The Ilizarov frame began to be used in western medicine during the 1980’s and by 1993 over 6000 cases had been performed in Europe.

Plain x-ray is one of the methods used to monitor the progress of patients fitted with an ilizarov frame.

The aim of this study is establish a pattern of healing over time in patients with the Ilizarov frame using plain x-ray films. This will improve understanding of the procedure, aid clinicians in deciding when frame removal is appropriate and provide a method of early detection should healing not be progressing appropriately.

This is a retrospective study looking at a series of 58 digitised anterior-posterior x-ray films of the tibia and fibula, taken at set time points post-operatively, from 17 patients fitted with an ilizarov frame (19 separate legs with ilizarov frames in total). Image J, an image analysis system, was used to measure pixel density from vertical slices down the centre of each fracture gap and at set intervals horizontally across the fracture gap. A mean pixel density value for each fracture gap was also calculated. The x-rays were standardised using a standard step wedge.

Promising preliminary results show pixel density to be greater towards the medial aspect of the tibia, but this difference in pixel value decreases with time. This suggests that calcification of the new bone occurs medially to laterally across the tibia. Full results will be available in April and aim to build a picture of the fracture gap at set time points post-operatively, showing a pattern of calcification in patients with the Ilizarov frame that will become a useful clinical tool for deciding time of frame removal as well as affording early knowledge of problems with the healing process.

The Ilizarov technique of distraction osteogenesis is becoming a more common way of treating complicated fractures. It has been shown that shear IFMs will delay bone healing whilst axial IFMs are beneficial to the bone healing. Therefore to measure IFMs in conditions of mobility will provide critical information for research and clinic diagnosis. Such data are not provided by static measurements. Traditionally the IFMs were measured by implanting transducers to the bone or using radiological methods. However, these methods are not suitable for either clinic utilization or measurement of IFMS when patients are doing movements which simulate their daily activities. We have designed a dynamic IFMs measuring device.

It includes a displacement transducer array, which is connected to the Ilizarov wires. This transducer array consists of 6 parallel linear displacement transducers, each of which is attached to the fixing wires of the fix-ator. This arrangement of transducers can fit into the configuration of Stewart Platform. The Reverse Stewart Platform algorithm was employed to calculate IFMs. Without measuring the bone fracture segments directly, the two segments were fitted into two planes virtually. By studying the relative movements of the two virtual planes, the algorithm transfers the relative movement to relative axial & shear translation, and relative bending & torsion rotation, between the two fracture segments. Wireless interface was used to transfer the displacement readings from the transducer array to the computer. This setup allows patient perform activities which represent their routine activities.

In laboratory studies, we found the error of this system to be related to the IFMs. For small movements around 100 micron, the absolute error was 50 micron, whereas for larger movements around 1 mm, the error was within 0.22mm.

This real time monitoring method will allow kinematical and kinetic studies on fracture patients treated with Ilizarov frame. Measurements obtained using this novel device will reflect the natural pattern of IFMs during the patients’ daily life. Since use of the device requires no additional pin, wire or operative procedure, it will be clinically applicable. The accurate real-time IFMs measurements will help elucidate the complex interplay between movement and bone formation.

Objective: To determine if the anatomical location of a tendon (hand or forearm) influences fibroblast function in the presence of physical forces.

Introduction Tendons are anatomical structures specialized to transmit high tensile loads from muscle to bone. When damaged, clinical recovery is slow and incomplete. Various authors have shown that application of tensile loading during recovery (such as in early active motion following hand flexor tendon repair) will accelerate the recovery of tensile strength. The mechanism is unknown and the optimum loading regime has not been quantitated. It is likely that similar influences are working in rheumatoid arthritis but there is clinical evidence that the response to applied load is very different. In this study a commercial system (Bio stretch) was used to apply different strain regimes to cells in culture, and then to assess the response by a series of quantitative methodologies

Materials Cells were obtained by the explant technique from tendons of the hand and forearm to generate confluent cultures. In this experiment fibroblasts cultured from intra-synovial tendons (Group 1)were compared with cultured fibroblasts of forearm tendons (Group II). We used the Biostretch Apparatus (ICCT Technologies Canada), to stretch fibroblasts in a gel foam (Helistat, Integra TM ) construct. The Biostretch apparatus uses a magnetic field to stretch cells within the gel foam. After seeding the gel foam pieces (1cm2) with a concentrated cell suspension (4 x105 cells/100 μlitre) , the apparatus was used at 40% stretch, with a burst time of 15 minutes and a rest time of 45 minutes at 37° C and 60 cycles a second for 24 hours. The experiment was performed in triplicate for both type of cells (Group I & II), with another group of cells serving as controls. At the end of 24 hours the BCA method was used to estimate Total Protein content while the Sircol method was used to determine Type 1 Collagen levels.

Results: Preliminary results indicate that there is a trend towards increased secretion of proteins and collagen in the stretched samples compared to the controls. Similarly the fibroblasts obtained from intra-synovial tendons seemed to produce more total protein and collagen as compared to the forearm. However both these observations failed to reach statistical significance.

Conclusions: Previous work (Evans CE et al. 2001) has shown no difference between collagen and protein production between flexor and extensor tendon, even under strain,. In this study the increased production of matrix proteins and collagen under the influence of physical strain may explain why flexor tendon injuries in the hand tend to heal with the formation of adhesions and poor functional results as compared with the forearm where the results tend to be uniformly better. However it must be stressed that these are preliminary results and further work will be required to provide definitive data.

Mechanical load is crucial to maintaining skeletal homeostasis, but the pathways involved in mecha-notransduction are still unclear. The OPG/RANK/ RANKL triumvirate has recently been implicated in bone homeostasis. These molecules, which are produced by the osteoblast (OPG and RANKL) and the macrophage/osteoclast (RANK), modulate osteoclastogenesis. We have previously shown that cyclical hydrostatic pressure influenced synthesis of various molecules by cultured human macrophages. These factors are important in osteoclastogenesis and bone resorption and have been linked to the development of aseptic loosening. We have also demonstrated that 1,25-dihydroxyvitamin D3 (1,25D3) influences macrophage response to pressure. For this study human macrophages were co-cultured with osteoblasts and subjected to cyclical hydrostatic pressure (34.5x10–3MPa [5.0 psi]) for up to five days, with or without 1,25D3 supplementation. Cells were immunostained for RANK and culture media were assayed for sRANKL and OPG using specific ELISAs. Immunostaining for RANK showed that macrophages subjected to pressure or 1,25D3 supplementation synthesised more RANK than controls. In addition, when exogenous 1,25D3 and hydrostatic pressure were administered simultaneously, immunostaining for RANK was more intense. There was a reciprocal relationship between OPG and sRANKL in co-cultures subjected to pressure. If pressure increased synthesis of sRANKL, OPG was decreased. In cultures where pressure decreased sRANKL, a corresponding increase in OPG was seen. In addition, samples from different individuals responded differently to pressure. The majority of cell populations responded to pressure by increasing OPG synthesis, compared to non-pressurised controls. These results demonstrate for the first time that the OPG/RANK/RANKL complex is sensitive to hydrostatic pressure and that 1,25-dihydroxyvitamin D3 might be involved in this response. These findings suggest a possible transduction mechanism for mechanical load in the skeleton, which has implications for future therapies for aseptic loosening and for skeletal abnormalities such as osteoporosis.

Aseptic loosening is a growing problem for orthopaedic surgeons and the importance of elevated hydrostatic pressure in its development in vivo is now well documented, but the mechanisms by which pressure could enhance loosening are unclear. We have demonstrated that hydrostatic pressures increased MP synthesis of cytokines, chemokines, PGE2 and M-CSF in vitro, all of which are implicated in bone resorption. 1,25-dihydroxy vitamin D3 (1,25D3) has a pivotal role in bone resorption. It stimulates osteoclastic bone resorption and formation, causes fusion of committed osteoclast precursor cells and activates mature osteoclasts in vitro. Under the correct conditions, macrophages (MP) have the ability to differentiate into osteoclasts. Research has shown that MP can synthesise 1,25D3 and changes in this synthesis occur during MP differentiation. We therefore examined how the application of hydrostatic pressure to MP in vitro influenced their synthesis of 1,25D3. In this study, normal human peripheral blood MP (5x105/ml) were cultured for 7 days then exposed to physiological pressure (34.5x10-3MPa) and/or UHMWPE particles (8mg/ml) and the effect on synthesis of 1,25D3 by endogenous 1a-hydroxylase (1aOHase) was studied. MP were incubated with H3-25, hydroxy vitamin D and 1,25D3 synthesis was analysed by HPLC. 1,25D3 synthesis was increased in cells under pressure by an average of 17% compared to static controls. In situ hybridisation (ISH) was used to demonstrate expression of 1aOHase. Image analysis showed a small increase in 1aOHase mRNA in response to pressure and to particles, and a larger increase to the two stimuli simultaneously. Expressed as % of maximum +Pressure + Particles 100%;+ Particles 59%; +Pressure 37%; No Stimulus < 0.1%. These results suggest that 1,25D3 may be one of the factors which stimulates osteoclastic bone resorption in aseptic loosening. As both these stimuli are likely to be present in vivo, such synthesis could further exacerbate loosening.

Background: Perioperative red cell salvage may be of use in cases where significant blood loss is likely. The purpose of this investigation was to see if its use in revision hip surgery led to a reduction in homologous blood transfusion requirement.

Methods: 48 patients were identified who had undergone revision hip surgery with the use of a Cell Saver device for perioperative autologous transfusion. Patients were individually matched to control patients who had undergone revision hip surgery without the Cell Saver. Patients were matched for age, sex and eight operative variables, which were chosen to indicate the type of revision surgery and possible level of blood loss, to ensure that the groups were comparable. Total homologous transfusion requirement in both groups was recorded as well as pre and post-operative haemoglobin levels.

Results: The groups were well matched for age, sex and operative variables. The total homologous transfusion requirement was significantly lower in the Cell Saver group than the control group (mean 2.6 v 6.4 units of packed cells respectively, p 0.0006). There was no difference in pre-operative haemoglobin between the groups but it was lower in the Cell Saver group post-operatively (Cell Saver 10.1g/dl v Control 10.6g/dl, p 0.06). There was no difference in length of operation.

Conclusions: Use of perioperative red cell salvage was associated with significantly lower homologous transfusion requirement. This is the first study looking at the use of perioperative red cell salvage in revision hip surgery with matching of patients on the basis of operative variables. A cost analysis shows that use of the Cell Saver has significant financial advantage in these patients.

Background: Perioperative red cell salvage may be of use in cases where significant blood loss is likely. The purpose of this investigation was to see if its use in revision hip surgery led to a reduction in homologous blood transfusion requirement.

Methods: 48 patients were identified who had undergone revision hip surgery with the use of a Cell Saver device for perioperative autologous transfusion. Patients were individually matched to control patients who had undergone revision hip surgery without the Cell Saver. Patients were matched for age, sex and eight operative variables ,which were chosen to indicate the type of revision surgery and possible level of blood loss, to ensure that the groups were comparable. Total homologous transfusion requirement in both groups was recorded as well as pre and post-operative haemoglobin levels.

Results: The groups were well matched for age, sex and operative variables. The total homologous transfusion requirement was significantly lower in the Cell Saver group than the control group (mean 2.6 v 6.4 units of packed cells respectively, p 0.0006). There was no difference in pre-operative haemoglobin between the groups but it was lower in the Cell Saver group post-operatively (Cell Saver 10.1g/dl v Control 10.6g/dl, p 0.06). There was no difference in length of operation.

Conclusions: Use of perioperative red cell salvage was associated with significantly lower homologous transfusion requirement. This is the first study looking at the use of perioperative red cell salvage in revision hip surgery with matching of patients on the basis of operative variables. A cost analysis shows that use of the Cell Saver has significant financial advantage in these patients.

Aims: This study investigates the use of novel autologous bone marrow plugs as a biological ÒmatrixÒ to support transgene expression following genetic modiþcation in vitro, and to deliver gene vectors to cartilage defects in vivo. Methods: Adenoviral vectors encoding marker genes (luciferase, green ßuorescent protein (GFP)) and bioactive genes (TGF-?) as well as genetically modiþed mesenchymalstem cells were used to characterize an autologous delivery system using clots of bone marrow aspirates in vitro, and within rabbit osteochondral defects in vivo. Results: Bone marrow clots were able to support expression of luciferase and TGF-? transgenes for up to 21d. In addition incubation of bone marrow clots with rTGF-? demonstrated, that the clots have chondrogenic potential, as evidenced by type II collagen and proteogly-can staining. Bone marrow clots seeded with cells genetically modiþed to express luciferase were able to support transgene expression following implantation into rabbit osteochondral defects for up to 14 days. Implanted clots were able to remain within the defects without þxation, and considerable integration with surrounding tissue was observed after 3 days. The bone marrow clots were also able to effectively localize transgene expression within the defects without leakage to surrounding tissue. Conclusion: These results demonstrate that genetically modiþed bone marrow plugs can support persistent transgene expression in vitro and within osteochondral defects in vivo. They provide an effective delivery system with chondrogenic potential.

Aims: This in vitro study investigates the use of Collagen/PRP Hydrogels as a biological matrix for containing genetically modified human ACL cells, and supporting transgene expression. Methods: Adenoviral vectors encoding marker genes (green fluorescent protein (GFP)) and bioactive) where used to infect cultured human ACL cells?genes (TGF- ex vivo. The cells were seeded in Collagen/PRP Hydrogels and maintained in culture. To expression over time, ELISA was performed at days 4, 8, 15, 23,?measure TGFand 29. GFP positive cells within the gel were viewed by fluorescence microscopy at the same time points. After 29 days, the cultures were fixed, sectioned and various sections were stained with H& E, toluidine blue to detect proteoglycans and by immunhistocemistry for collagen type I and II. Results: Collagen/PRP Hydrogels were transgenes for up to 29 days.?able to support expression of GFP and TGF- expressing gel/cell constructs produced an abundant?Compared to controls, TGF- amount of type I collagen, consistent with the ligament phenotype and appeared more cellular. Little or no proteoglycan staining was observed in either group. Conclusion: These results demonstrate that genetically modified human ACL cells can support persistent transgene expression in vitro, sufficient to stimulate growth of ligamentlike tissue within a Collagen/PRP Hydrogel. The high levels of transgene expression suggest that the Collagen/PRP Hydrogel can function as an effective gene delivery system for tendon repair in vivo.

Objective: To determine if (a) inflammatory mediators are present in herniated intervertebral discs and (b) if their presence correlates with inflammation of nerve roots or symptoms.

Design: Inflammation was assessed with gadolinium enhancement of MRI. Neurological compromise was measured. Disc tissue was examined for inflammatory mediators IL-1α and β, IL-6, MCP-1, TSG-6, iNOS, TNFα and thromboxane.

Patients: Sixty-five discs were removed from 64 patients undergoing surgery for disc prolapse.

Outcome measures: We developed (i) an MRI score to assess inflammation radiologically prior to surgery (n=28, mean 4.9±6.8 days), (ii) a Surgical Score to assess inflammation of the nerve roots at surgery (n=44), (iii) a Clinical Score to determine pain, disability and neurological compromise (n=17) and (iv) a Mediator Score to reflect the number and amount of inflammatory mediators present (n=20).

Results: Thirty percent of the prolapses in this study were extrusions, 19% sequestrations and 51% protrusions. Sixteen of the 28 patients with gadolinium had nerve root enhancement (86% of the extrusions, 57% of sequestrations, and 43% of protrusions), whilst 19 had enhancement of or around the disc herniation itself (71% of the extrusions, 86% of sequestrations and 57% of protrusions). The Mediator Scores were highest for the sequestrations (as was the Surgical Score) and lowest for the protrusions, but extruded discs had most IL-1α and β, IL-6, TNFα and thromboxane. Extruded discs had the highest Clinical Score and sequestrated the lowest.

Conclusions: Mediators produced in prolapsed disc appear to play an important role in inflammation of adjacent tissue and nerve roots. The type of mediator present and proximity of the prolapse to the nerve root may be the important factors in determining which pro-lapses are the most painful.

Unlike most other tumours, myeloma causes bone destruction without an osteoblastic reaction; we tried to assess whether myeloma secretes a humoral factor that inhibits osteoblasts. Human bone-derived cells were either co-cultured with myeloma cells, or cultured in medium conditioned by myeloma cells. Bone-derived cell growth was measured by cell counts and by uptake of tritiated thymidine (3H-Tdr); growth was inhibited when cultured in medium conditioned by myeloma cells and some inhibition was seen when the bone-derived cells were co-cultured with myeloma cells. The inhibiting effect was dose-dependent and also dependent upon the density of the myeloma cells conditioning the medium. The results of our study suggest that myeloma secretes an osteoblast inhibiting factor of less than 50,000 Dalton molecular weight.

Ferrography is a technique for analysing wear by means of the magnetic separation of wear particles. To evaluate its application in human joints, the results of the ferrographic analysis of saline washings of symptomatic human knees were compared with the results of the arthroscopic examination of the same knees. Ferrography was found to be an extremely sensitive monitor of articular erosion, with a resolution far greater than that of arthroscopy. This was particularly apparent with knees suffering from a torn anterior cruciate ligament: arthroscopy detected no damage to the cartilaginous surfaces whereas ferrography detected a substantial level of "microdamage". The spectrum of wear particles showed qualitative and quantitative alterations depending upon the condition of the knee. Ferrography thus holds much promise as a potential differential diagnostic technique of great sensitivity, with particular relevance to the very early changes which precede clinical symptoms. Study of wear particles is also justified by evidence indicating an active role in the pathophysiological progression of arthritis.