MiRNAs perform gene regulation that can target approximately 60% of human protein coding genes. Along with many cellular processes, miRNAs have been implicated in stem cell differentiation. Osterix (Osx), which is inhibited by mir-31, is required by MSCs for early osteoblast differentiation resulting in bone formation further downstream. We used antagomir functionalised gold nanoparticles (AuNPs) to block mir-31, which resulted in upregulation of Osx in pre-osteoblastic MG63 cells and human mesenchymal stem cells (MSCs).

We used MG63 pre-osteoblastic cell line and human MSCs. Cytotoxicity of AuNPs was assessed by MTT, and cellular uptake of AuNPs was verified by TEM and ICP-MS. Osx RNA levels were determined by Fluidigm analysis and protein expression by In Cell Western analysis.

Antagomir-functionalised AuNPs were incubated with cells for an initial 48 hours. (1) No cytotoxic effects were noted in either cell type. (2) Fluidigm analysis identified a varied gene response to antagomir delivery in both cell types, with MSCs recording a reduction of stem cell marker genes nestin, alcam, CD63, and CD44 at day 5 (indicating differentiation). (3) Osx protein levels were increased in both cell types after 48 hour incubation. (4) Downstream MSC analysis demonstrated accelerated osteogenesis at week 3 and 5 (verified by osteocalcin nodule formation) following 48 hour AuNP incubation.

RNA analysis in both cell types suggested a shift away from proliferation towards osteoblastic differentiation. This was supported by Osx protein expression, which was increased in both MG63 cells and MSCs. Finally, an increase in the late osteogenic marker (osteocalcin) was verified at weeks 3 and 5 in MSCs after AuNP incubation for 48 hours. These results collectively infer successful delivery of mir-31 antagomirs, which are blocking mir-31-mediated suppression of Osx, resulting in an early increase in Osx, which accelerates MSC osteogenesis downstream.

Osteoporosis is characterised by an uncoupling of bone formation and resorption resulting in net resorption. Stem cells derived from bone marrow in osteoporotic patients typically contain more adipocytes. Intermittent Parathyroid hormone (iPTH), has been shown to cause the preferential differentiation of mesenchymal stem cells (MSCs) to osteoblasts. We isolated rat bone marrow derived MSCs, investigating the effect of iPTH on adipocyte differentiation.

MSCs were harvested from the femora of 6–10week oldWT rats and cultured to induce adipogenesis for 21 days. Subsequently, cells were continually cultured in adipogenic media, osteogenic media or in osteogenic media supplemented with PTH 1–34 either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Oil Red O used to assess intracellular microdroplet formation. A student t-test was used to analyse results, and a p value<0.05 considered significant.

Quantitatively measurements of Alizarin Red staining significantly increased in all adipocytes grown in osteogenic media compared to the cells continually cultured in adipogenic media. Calcium phosphate deposition continued to increase significantly in these groups up to day 14. At day 14, Alizarin Red staining from cells cultured in iPTH were significantly higher than osteogenic media alone.

ALP expression was significantly higher for cells cultured in osteogenic media and iPTH compared to adipogenic media at days 3–14. Expression peaked at day 7, at this timepoint cells cultured in iPTH expressed significantly more ALP than other groups (Figure 2). Oil Red O measurements were significantly reduced from days 7–14 for all osteogenic groups, this significance was greatest for the iPTH group at day 7.

iPTH increased the transdifferentiation of adipocytes derived from MSCs into osteoblasts, this effect was most significant after 7 days. Ultimately, the role of iPTH on adipocytes may lead to improved bone formation with many orthopaedic applications.

Hematopoietic stem cells (HSCs) reside within a specialised niche area in the bone marrow (BM). They have tremendous clinical relevance, although HSC expansion and culture ex vivo is not currently possible, reducing BM transplant success. This project expands a novel 3D MSC niche model developed in our lab to include HSCs.

MSCs were loaded with green fluorescent magnetic iron oxide (FeO₃) nanoparticles (200 nm diameter) at a concentration of 0.1 mg ml⁻¹, and incubated for 30 min over a magnet to enhance cellular uptake. The cells were washed, detached and resuspended, then transferred to a plate with magnets above. Spheroids formed within hours and were implanted into 2 mg ml⁻¹ collagen gel. HSCs were loaded with nanoparticles via incubation with suspension, and then introduced to the gel containing the spheroid. Immunostaining, BrdU and Calcein/ ethidium homodimer viability assays were performed to characterise the cells.

Cells in both monolayers and spheroids remain viable up to 7 days in culture. MSCs in monolayers and spheroids were stained with antibodies for: STRO-1, an MSC marker; SDF-1 (CXCL-12), a secreted HSC homing factor; and nestin, a marker for HSC-supportive MSCs in vitro. MSCs in spheroids retain a higher level of expression of all three for 7 days compared to MSCs in monolayers. BrdU assay results show that the MSCs are more quiescent in spheroids compared to monolayers.

Proof of principle studies are promising for the success of the proposed niche model. MSCs express a higher level of MSC markers and retain quiescence when they are in spheroids as compared to monolayers. They also express a higher level of HSC niche factor SDF-1α, which facilitates HSC migration and retention.

Mesenchymal stem cells (MSCs) are usually believed to be immune-privileged. However, immunogenic MSCs were also reported. We hypothesize that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immunogenicity. Our goal is to discover immune-privileged stem cells for universal allogenic MSCs transplantation.

Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. The proliferation rate of each clonal culture and mixed clonal culture were measured. The tri-differentiation potential of the clonal cultures was compared, as well as with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. Mixed leucocyte reaction (MLR) were also performed to investigate immunogenicity.

Tri-differentiation was confirmed in all isolates. All clonal cultures revealed significant different morphology and proliferation rates, compared with each other and mixed cultures. All clonal cultures showed different surface markers, inclusive of MHC antigens. One clone from ADMSCs showed lack of MHC antigens. Our MLR and MHC staining disclosed variety of immune properties.

All clones tri-differentiated which indicated a degree of ‘stemness’. MSCs are generally believed not to express MHC II, resulting in immune-privileged. Our results confirmed our hypothesis because clonal cultures isolated from different origins of same animal show differences in morphology, proliferation rate, and surface marker presentation. Individual immune differences highlighted through single-cell clonal cultures may be crucial to find universal immune-privileged MSCs as universal allogeneic donor.

Material-based strategies seek to engineer synthetic microenvironments that mimic the characteristics of physiological extracellular matrices for applications in regenerative therapies, including bone repair and regeneration. In our group, we identified a specific chemistry, poly(ethyl acrylate) (PEA), able to induce the organization of fibronectin (FN), upon adsorption of the protein, into fibrillar networks similar to the physiological ones, leading to enhanced cellular response, in terms of cell adhesion and differentiation. In this work, we exploit these FN networks to capture and present growth factors (GF) in combination with the integrin binding domain of FN during bone tissue healing.

Fibrillar conformation of FN adsorbed on PEA favors the simultaneous availability of the GF binding domain (FNIII12–14) next to the integrin binding region (FNIII9–10), compared to poly(methyl acrylate) (PMA), a material with similar chemistry, where FN adopts a globular conformation. The combined exposure of specific adhesive sequences recognized by integrins and GF binding domains was found to improve the osteogenic differentiation of mesenchymal stem cells. A higher expression of bone proteins was found when BMP2 is bound or sequestered on the material surface versus its administration in the culture media in vitro. The potential of this system as recruiter of GFs was also investigated in a critical-size bone segmental defect in mouse. The synergistic integrin-GF signalling, induced by fibrillar FN, promoted bone formation in vivo with lower BMP2 doses than current technologies. Furthermore, we optimized the system for its potential use in translational research, seeking to address the clinical need of using biocompatible and biodegradable material implants. Polycaprolactone scaffolds were synthesized and coated with a thin layer of plasma- polymerized PEA that recruits and efficiently presents GF during healing of critical size defects.

The material-driven FN fibrillogenesis provides a new strategy to efficiently reduce the GF doses administrated in bone regenerative therapies.

Control of stem cell fate and function is critical for clinical and academic work. By combining surface chemistry-driven extracellular matrix (ECM) assembly with mesenchymal stem cells (MSCs) we are developing a system which can be used to regulate the behaviour of MSCs. The conformation of the ECM glycoprotein fibronectin (Fn) is different when adsorbed onto poly methylacrylate (PMA) where it is globular, and on poly ethylacrylate (PEA) where it forms a physiologically-similar network^[1] (Fig. 1). Using these polymers to govern Fn conformation, we are developing a 3D system incorporating MSC-responsive growth factors (GFs) and bone marrow MSCs capable of regulating MSC behaviour.

Toluene-dissolved PMA and PEA were spin coated onto glass coverslips before solvent extraction in vacuo and UV sterilisation. 20 mg ml⁻¹ human plasma FN was adsorbed onto the surfaces followed by 25 ng ml⁻¹ recombinant human BMP2/VEGF. FN conformations were characterised by atomic force microscopy (AFM). A collagen hydrogel was placed above the substrate. Adult human bone marrow STRO-1+ were cultured on the substrates for 3 weeks in supplemented DMEM. Expression of MSC stemness and HSC maintenance factors were analysed by In-Cell Western assay.

To establish the best combination of polymer/FN/GF, MSC stemness markers (ALCAM, NESTIN and STRO1), osteogenic differentiation markers (OCN and OPN) and bone marrow markers (SCF and VCAM1) were measured in MSCs cultured for 3-weeks on substrates. OCN, SCF, and VCAM1 expression was enhanced across all combinations compared to glass control, while for ALCAM/STRO1/NESTIN and OPN, PEA combinations enhanced their expression. PEA + FN + VEGF appeared to be system best suited to maintaining MSC stemness and supporting expression of osteogenesis markers and bone marrow markers.

We have shown that MSCs maintain their stem cells state and express high levels of SCF and VCAM-1 when cultured on PEA with adsorbed Fn and VEGF or BMP2. Next stages of this work will use PCR to verify results and analyse expression of other MSC markers to develop a role for these synthetic polymers as biomaterials.

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable.

To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhesion was quantified at 1, 2, 6, 12 and 24 hours by live/dead counts and biofilm mass quantified by crystal violet staining after 24, 48, 72 and 96 hours culture. To elucidate the mechanisms of action of LPA, proteomic analysis of adhered bacteria was performed using SDS-PAGE and Western blots.

500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability.

LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life.

We have developed precision-engineered strontium eluting nanopatterned surfaces. Nanotopography has been shown to increase osteoblast differentiation, and strontium is an element similar to calcium, which has been proven to increase new bone formation and mineralization. This combination has great potential merit in fusion surgery and arthroplasty, as well as potential to reduce osteoporosis. However, osteoclast mediated osteolysis is responsible for the aseptic failure of implanted biomaterials, and there is a paucity of literature regarding osteoclast response to nanoscale surfaces. Furthermore, imbalance in osteoclast/osteoblast resorption is responsible for osteoporosis, a major healthcare burden. We aimed to assess the affect of strontium elution nanopatterned surfaces on osteoblast and osteoclast differentiation.

We developed a novel human osteoblast/osteoclast co-culture system without extraneous supplementation to closely represent the in vivo environment. We assessed the surfaces using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR).

In complex co-culture significantly increased osteoblast differentiation and bone formation was noted on the strontium eluting, nanopatterned and nanopatterned strontium eluting surfaces, suggesting improved osteointegration. There was a reduction in macrophage attachment on these surfaces as well, suggesting specific anti-osteoclastogenic properties of this surface.

Our results show that osteoblast and osteoclast differentiation can be controlled through use of nanopatterned and strontium eluting surface features, with significant bone formation seen on these uniquely designed surfaces.

Long-term survival of massive prostheses used to treat bone cancers is associated with extra-cortical bone growth and osteointegration into a grooved hydroxyapatite coated collar positioned adjacent to the transection site on the implant shaft [1]. The survivorship at 10 years reduces from 98% to 75% where osteointegration of the shaft does not occur. Although current finite element (FE) methods successfully model bone adaption, optimisation of adventitious new bone growth and osteointegration is difficult to predict. There is thus a need to improve existing FE models by including biological processes of osteoconduction and osteoinduction.

The principal bone adaptation criteria is based on the standard strain-energy remodeling algorithm, where the rate of remodeling is controlled by the difference in the stimulus against the reference value [3]. The additional concept of bone connectivity was introduced, to limit bone growth to neighbouring elements (cells) adjoining existing bone elements. The algorithm was developed on a cylindrical model before it was used on an ovine model.

The geometry and material properties from two ovine tibiae were obtained from computed tomography (CT) scans and used to develop FE models of the tibiae implanted with a grooved collar. The bones were assigned inhomogeneous material properties based on the CT grey values and typical ovine walking load conditions were applied. The FE results show a region of bone tissue growth below the implanted collar and a small amount of osteointegration with the implant, which is in good agreement to clinical results. Some histological results suggest that further bone growth is possible and potential improvements to the model will be discussed. In summary, by including an algorithm that describes osteoconduction, adventitious bone growth can be predicted.

Poly-lactic acid (PLA) scaffolds are widely used in bone tissue engineering. The introduction of 3D printing has greatly increased the ability for tailoring different geometrical designs of these scaffolds for improved cellular attachment, growth and differentiation. This study aimed to investigate the effect of PLA fibre angle in 3D printed PLA scaffolds on hDPSC attachment and growth in vitro.

Two types of PLA scaffolds were prepared via 3D printing containing fibres angled at either 45° or 90°. hDPSCs (P4, 2*10⁵ cells per scaffold) were statically seeded for 4 hours on to the scaffolds (7×3.5×3 mm³, n=3). Cellular attachment was checked using fluorescence microscopy and the number of unattached cells was counted using a haemocytometer (HCM). The cell-scaffold constructs were then cultured in osteogenic medium for up to 5 weeks. ALP staining and SEM were performed for one construct from each group at week 3. Cellular viability was determined using CMFDA/EHD1 live/dead labelling at week 4. After 5 weeks, constructs were processed for histology.

Fluorescence micrographs showed high numbers of hDPSCs attached to scaffold surfaces in both groups after seeding irrespective of fibre angle. However, HCM cell count revealed that the 45° angled PLA scaffolds had significantly greater cell attachment compared to the 90° angled PLA group (p<0.0001). After 3 weeks in osteogenic culture, both types of construct showed strong ALP staining. SEM showed that in the 45° angled PLA group, almost all macro-pores were fully closed with newly formed cell sheets. In comparison, in the 90° angled group, most of the macro-pores remained open although a limited amount of cellular bridging was present. SEM also detected crystal deposits in different areas within the cell sheets for both construct groups. Most hDPSCs were alive in both groups at week 4 of culture with few dead cells present. After 5 weeks, histology showed marked cellular growth and new matrix formation, with detectable Van Kossa +ve crystal deposits in different areas within all constructs irrespective of PLA fibre angle.

This study showed that 45° angled PLA 3D printed scaffolds enhanced hDPSC attachment and cellular bridging, which may help to rapidly close the macro-pores within the scaffold compared to the 90° angled group. This illustrates the potential of 45° angled 3D printed PLA scaffolds as good candidates for bone tissue engineering.

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.

Tissue expansion is a technique used by plastic and restorative surgeons to cause the body to grow additional skin, bone or other tissues. For example, distraction osteogenesis has been widely applied in lower limb surgery (trauma / congenital), and congenital upper limb reconstruction (e.g. radial dysplasia). This complex and tightly regulated expansion process can thus far only be optimised by long-term animal or human experimentation.

Here the intent is to develop an in vitro model of tissue expansion that will allow to both optimise the extension regime (µm/h, continuous/ intermittent) and investigate using proteomic techniques which molecular pathways are involved in its regulation. Cells cultured onto sheets of polymer (PCL) can be stretched at very low, adjustable speeds, using a stepper motor and various 3D printed and laser cut designs. The system utilises plastic flow of the polymer, enabling the material to stay extended upon strain being released.

Tensile tests have displayed the plastic behaviour of the polymer sheet when stretched, and digital image correlation (DIC) has been used to analyse homogeneity of the strain field. Further analysis involving nuclear localisation of yes-associated protein (YAP) aims to link cell response to this strain field.

Nuclear orientation analysis has demonstrated a morphological response to strain (1 mm/day) in comparison to not being stretched, and this is in the process of being linked to nanoscale changes of the substrate (using atomic force microscopy) during the stretching regime. Future work will identify how strain is affecting the cell cycle, before a mass tagging approach is used to identify protein changes induced by strain.

Osteoarthritis (OA) is no longer considered a cartilage-centric disease with remodelling of other joint tissues now recognized. While understudied, entheseal pathology is considered a secondary OA feature. A pivotal role for proteinase-activated receptor 2 (PAR2) in OA has been demonstrated previously in cartilage and subchondral bone at early time points, however the entheseal role of PAR2 has not been reported.

OA was induced by destabilization of the medial meniscus (DMM) in wild type (WT) and PAR2 deficient (KO) animals. At 4 weeks and one year post surgery, knee joints were harvested for histological analysis. Medial collateral ligament (MCL) width was measured by 2D planimetry analysis. Immunohistochemistry was used to characterize the MCL and anterior cruciate ligament (ACL). Data were expressed as mean±SEM (n=4–6/group) and analysed using Student's t-test, with p<0.05 as the criterion of significance.

MCL width increased between 4 weeks and 1 year in WT DMM (0.24 ±0.07 vs 0.40 ±0.008mm respectively, p<0.001). Interestingly, a significant reduction in MCL was observed in KO compared with WT at 1 year (0.23 ±0.005 vs 0.40 ±0.008mm respectively, p <0.001) post-DMM. Further characterization of DMM WT MCL and ACL at 4 weeks showed the presence of F4/80⁺ cells in addition to IL-33 and histamine. At one year post-surgery, a cellular infiltrate was observed in MCL DMM WT but absent in KO mice. Histological evaluation revealed an absence of F4/80⁺ cells but the presence of a PAR2⁺ population, subsequently identified as hypertrophic-like chondrocytes (RUNX2) and chondrocytes-like cells (SOX9).

Deletion of PAR2 affords long-term protection against ligament remodelling and demonstrates a critical role for this receptor in both OA joint pathology and ligament injuries. While PAR2 appears to be a credible therapeutic target in OA entheseal pathology, further understanding of the molecular mechanism regulated by this receptor will be required.

Entheses are the anchorage sites of tendons to bones in the musculoskeletal system. They have a unique microanatomy that allow smooth transfer of mechanical load through tendon to bone. However, entheses are prone to injury due to their small surface area^1,2. The overall success rate of the current gold standard treatment (directly attaching the tendon to bone) is small^3,4. Consequently, the aim of this study was to evaluate different hydrogels and their suitability for developing an in-vitro co-culture system to manufacture 3D tissue interfaces.

To create a 3D in-vitro tissue interface, half-well plugs were created by pouring silicone in wells of a 24-well plate. When set, it was cut into halves to be used as half-well plugs, blocking one side of a culture well. A tendon-cell-encapsulated hydrogel was poured into the exposed half and, when set, the plug was removed and a bone-cell-encapsulated gel was added. Cells were fluorescently labelled to enable identification of cell types under fluorescent microscopy (Tendon – green, bone – red). The suitability of different hydrogels to form an in vitro tissue interface was evaluated: fibrin, agarose and gellan.

This study demonstrates that 3D co-cultures can be manufactured in-vitro. The novel system enabled the culture of two cell types (bone/tendon) in direct contact, creating an in-vitro interface. In addition, this study shows that fibrin gel supports cell morphology, while both cell types failed to show normal morphology in agarose and gellan. Further studies evaluating cell viability in these hydrogels are currently underway.

Acellular porcine super flexor tendon (pSFT) offers a promising solution to replacement of damaged anterior cruciate ligament [1]. It is desirable to package and terminally sterilise the acellular grafts to eliminate any possible harmful pathogens. However, irradiation techniques can damage the collagen ultra-structure and consequently reduce the mechanical properties [2]. The aims of this study were to investigate the effects of irradiation sterilisation of varying dosages on the biomechanical properties of the acellular pSFT.

Tendons were decellularised using a previously established protocol [1] and subjected to irradiation sterilisation using either 30 kGy gamma, 55 kGy gamma, 34 kGy E-beam, 15 kGy gamma, 15 kGy E-beam and (15+15) kGy E-beam (fractionated dose). Specimens then underwent stress relaxation and strength testing at 0 and 12 months post sterilisation to determine whether any effect on these properties was progressive. For stress relaxation testing, specimens were analysed using a Maxwell-Wiechert model. For strength testing, the ultimate tensile strength, Young's modulus and failure strain were assessed.

Significant differences were found which demonstrated that all irradiation treatments had an effect on the time-independent and time-dependent viscoelastic properties of irradiated tendons compared to per-acetic acid only treated controls. Interestingly, no significant differences were found between the irradiated groups. Similar trends were found for the strength testing properties. No significant differences were found between groups at 0 and 12 months.

Tendons retained sufficient biomechanical properties following sterilisation, however it was notable that there were no significant differences between the irradiated groups, as it was believed higher dosages would lead to a greater reduction in the mechanical properties. The changes observed were not altered further after 12 months storage, indicating the acellular pSFT graft has a stable shelf-life.

Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes.

Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA.

Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling.

The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders.

Currently available fracture fixation devices that were originally developed for healthy bone are often not effective for patients with osteoporosis. Resulting outcomes are unsatisfactory, with longer recovery times, often requiring re-surgery for failed cases. One major issue is the design of bone screws, which can loosen or pull-out from osteoporotic bone. Design improvements are possible, but the development of new screws is a lengthy and expensive process due to the manufacture of the complex geometry involved. The aim of this research was to validate our currently available 3D printing technology in the design, manufacture and testing of screws.

Three standard wood screw designs were reverse-engineered using computational modelling and then fabricated in polymeric resin using 3D rapid prototyping on a Stereolithography (SLA) machine. The original metal screws and the 3D screws (n=5 of each) were then inserted into a synthetic bone block (Sawbones, PCF5) representing the mechanical properties of severely osteoporotic cancellous bone. Pull-out tests were conducted in accordance with ASTM 543-13.

The three metal screws exhibited pull-out strengths of 125, 74 and 118 N respectively. The 3D printed screws by comparison showed pull-out strengths approximately 15–20 % lower than their metal counterparts. However, when the results were normalised to the material tested, showing the relative changes to the first design, the pattern of results in the metal and 3D printed groups were almost identical (within 3 % of each other), showing excellent correlation.

This study is the first to show that 3D Rapid Prototyping can be used in the pre-clinical testing of orthopaedic screws. The methodology provides a cheaper, faster development process for screws, allowing huge scope for development and improvement. Future work will include expanding the study to include more screw configurations as well as testing in higher density foams to compare performance in healthier bone.

A number of advantages of unicondylar arthroplasty (UKA) over total knee arthroplasty in patients presenting osteoarthritis in only a single compartment have been identified in the literature. However, accurate implant positioning and alignment targets, which have been shown to significantly affect outcomes, are routinely missed by conventional techniques. Computer Assisted Orthopaedic Surgery (CAOS) has demonstrated its ability to improve implant accuracy, reducing outliers. Despite this, existing commercial systems have seen extremely limited adoption. Survey indicates the bulk, cost, and complexity of existing systems as inhibitive characteristics. We present a concept system based upon small scale head mounted tracking and augmented reality guidance intended to mitigate these factors.

A visible-spectrum stereoscopic system, able to track multiple fiducial markers to 6DoF via photogrammetry and perform semi-active speed constrained resection, was combined with a head mounted display, to provide a video-see-through augmented reality system. The accuracy of this system was investigated by probing 180 points upon a 110×110×50 mm known geometry and performing controlled resection upon a 60×60×15 mm bone phantom guided by an overlaid augmented resection guide that updated in real-time.

The system produced an RMS probing accuracy and precision of 0.55±0.04 and 0.10±0.01 mm, respectively. Controlled resection resulted in an absolute resection error of 0.34±0.04 mm with a general trend of over-resection of 0.10±0.07 mm.

The system was able to achieve the sub-millimetre accuracy considered necessary to successfully position unicondylar knee implants. Several refinements of the system, such as pose filtering, are expected to increase the functional volume over which this accuracy is obtained. The presented system improves upon several objections to existing commercial CAOS UKA systems, and shows great potential both within surgery itself and its training. Furthermore, it is suggested the system could be readily extended to additional orthopaedic procedures requiring accurate and intuitive guidance.

3D imaging is commonly employed in the surgical planning and management of bony deformity. The advent of desktop 3D printing now allows rapid in-house production of specific anatomical models to facilitate surgical planning. The aim of this pilot study was to evaluate the feasibility of creating 3D printed models in a university hospital setting.

For requested cases of interest, CT DICOM images on the local NHS Picture Archive System were anonymised and transferred. Images were then segmented into 3D models of the bones, cleaned to remove artefacts, and orientated for printing with preservation of the regions of interest. The models were printed in polylactic acid (PLA), a biodegradable thermoplastic, on the CubeX Duo 3D printer.

PLA models were produced for 4 clinical cases; a complex forearm deformity as a result of malunited childhood fracture, a pelvic discontinuity with severe acetabular deficiency following explantation of an infected total hip replacement, a chronically dislocated radial head causing complex elbow deformity as a result of a severe skeletal dysplasia, and a preoperative model of a deficient proximal tibia as a result of a severe tibia fracture. The models materially influenced clinical decision making, surgical intervention planning and required equipment. In the case of forearm an articulating model was constructed allowing the site of impingement between radius and ulnar to be identified, an osteotomy was practiced on multiple models allowing elimination of the block to supination. This has not previously been described in literature. The acetabulum model allowed pre-contouring of a posterior column plate which was then sterilised and eliminated a time consuming intraoperative step.

While once specialist and expensive, in house 3D printing is now economically viable and a helpful tool in the management of complex patients.

Chondrocytes are essential to the maintenance of articular cartilage and it is thought that chondrocyte death occurs early in septic arthritis. Understanding the causes of chondrocyte death will allow the development of chondroprotective strategies to improve long-term outcomes following septic arthritis.

We utilised a murine model of septic arthritis using intra-articular injection of 10µL of a 10⁷ concentration of S. aureus suspended in PBS. Seventy-five adult male C57/Bl6 mice were randomised to receive injection of either S. aurues 8325-4 (a wild-type of S. aurues capable of alpha toxin production), DU1090 (an isogenic mutant of 8325-4 that is identical to 8325-4 other than being incapable of producing alpha toxin) or a PBS control. Establishment of septic arthritis was confirmed through gait changes (5 mice/group), limb swelling and histological changes (10 mice/group). 10 animals from each group were sacrificed at 48 hours and the injected knee joints were dissected before being stained with CFMDA (labelling live chondrocytes green) and PI (labelling dead chondrocytes red). The samples were imaged using a confocal laser scanning microscope and the percentage of chondrocyte death was calculated.

Mice injected with S. aureus 8325-4 or DU1090 developed septic arthritis with evidence of weight loss, limb swelling and gait changes whereas these were absent in the control group. There was a significantly higher level of chondrocyte death in the group infected with 8325-4 (2.7% chondrocyte viability) when compared to DU1090 (73.9% chondrocyte viability) and PBS injected mice (95% chondrocyte viability). One-Way ANOVA revealed that the difference between each group was statistically different (p < 0.05).

Alpha toxin is the major damaging toxin in S. aurues septic arthritis. Any adverse effect of the immune system is negligible in comparison. Development of treatments counteracting the effect of alpha toxin is required.

Demineralised dentine matrix (DDM) contains a myriad of growth factors and matrix proteoglycans, the bioactivity of which can utilised in dental restorations and bone augmentations. This study aimed to develop a novel antimicrobial, bioactive dental cement to promote reparative dentinogenesis and prevent infections, improving the longevity of current dental restorations.

Nanocarriers containing DDM (extracted from non-carious dentine; 1–100 μg/mL), and triclosan (300 μg/mL) were made. Human dental pulp stem cells (hDPSCs) were treated with DDM nanocarriers (10 ng/mL-100 μg/mL) for 3, 9, 21 and 35 days. Cell proliferation and viability were assessed by cell counts, Caspase-Glo 3/7 (Promega) and MTT assays. qRT-PCR was used to examine the expression of osteogenic markers runx2 and osteocalcin at days 3, 9 and 21. A transwell chemotaxis/ migration assay was used to assess the ability of DDM nanoparticles to recruit hDPSC progenitors. Triclosan nanocarriers were tested using growth curves and zones of inhibitions for S. Anginosus and E. Faecalis. SEM and biomechanical testing was carried out on Vitremer (Henry Schein) dental cements containing loaded and empty nanocarriers.

DDM nanocarriers were able to significantly recruit hDPSCs and induce the expression of osteogenic markers in hDPSCs after 9 days. DDM Nanocarriers had no effect on cell proliferation or survival. Triclosan nanocarriers were able to inhibit the growth of S. Anginosus and E. Faecalis. Nanocarriers had limited effect on the biomechanical integrity of Vitremer cements.

Nanocarriers successfully delivered DDM to hDPSC, promoting their in vitro recruitment and osteogenic differentiation, and triclosan to endodontic bacteria inhibiting their growth. The nanocarriers were incorporated into cements with minimal physical artefacts, therefore a novel antimicrobial, bioactive dental cement was produced, which could be a useful tool for dental tissue engineering.

Healthcare associated infections (HAI) pose a major threat to patients admitted to hospitals, and infection rates following orthopaedic arthroplasty surgery are as high as 4%, while the infection rates are even higher after revision surgery. 405 nm High-Intensity Narrow Spectrum (HINS) light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in orthopaedic surgery.

Cultured rat osteoblasts were exposed to 405 nm light to investigate if bactericidal doses of light could be used safely in the presence of mammalian cells. Cell viability was measured by MTT reduction and microscopy techniques, function by alkaline phosphatase activity, and proliferation by the BrdU assay. Exposures of up to a dose of 36 J/cm² had no significant effect on osteoblast cell viability, whilst exposure of a variety of clinically relevant bacteria, to 36 J/cm² resulted in up to 100% kill. Exposure to a higher dose of 54 J/cm² significantly affected the osteoblast cell viability, indicating dose dependency.

Work also demonstrated that 405 nm light exposure induces reactive oxygen species (ROS) production in both mammalian and bacterial cells, as shown by fluorescence generated from 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate dye. The mammalian cells were significantly protected from dying at 54 J/cm² by catalase, which detoxifies H₂O₂. Bacterial cells were significantly protected by sodium pyruvate (H₂O₂ scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (·OH scavenger), catalase) at 162 and 324 J/cm². Thus the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria is likely oxidative stress involving predominantly H₂O₂ generation, with other ROS contributing to the damage.

Additional work describing the potential for incorporation of this antimicrobial light within operating theatre lighting systems will also be discussed, and this, coupled with the cell viability and cytotoxicity results, suggests that 405 nm light could have great potential for continual patient safe decontamination during orthopaedic replacement surgeries and thereby reduce the incidence of infections.

One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus Aureus. Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface.

Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion on nanofabricated materials. We hypothesise that surface nanotopography impacts the differential ability of staphylococci species to adhere via altered metabolomics and may reduce orthopaedic implant infection rate.

Bacteria were grown and growth conditions optimised. Polystyrene and titanium (Ti) nanosurfaces were studied. The polystyrene surfaces had different nanopit arrays, while the Ti surfaces expressed different nanowire structures. Adhesion analysis was performed using fluorescence imaging, quantitative PCR and bacterial percentage coverage calculations. Further substitution with ‘heavy’ labelled glucose into growth medium allowed for bacterial metabolomic analysis and identification of any up-regulated metabolites and pathways.

Our data demonstrates reduced bacterial adhesion on specific nanopit polystyrene arrays, while nanowired titanium showed increased bacterial adhesion following qPCR (P<0.05) and percentage coverage calculations (P<0.001). Further metabolomic analysis identified significantly increased intensity counts of specific metabolites (Pyruvate, Aspartate, Alanine and Carbamoyl aspartate).

Our study shows that by altering nanotopography, bacterial adhesion and therefore biofilm formation can be affected. Specific nanopatterned surfaces may reduce implant infection associated morbidity and mortality. The identification of metabolic pathways involved in adhesion may allow for a targeted approach to biofilm eradication in S. aureus. This is of significant benefit to both the patient and the surgeon, and may well extend far beyond the realms of orthopaedics.

Nanotopographical cues on Ti surfaces have been shown to elicit different cell responses such as differentiation and selective growth. Bone remodelling is a continuous process requiring specific cues for optimal bone growth and implant fixation. In addition, the prevention of biofilm formation on surgical implants is a major challenge. We have identified nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. We used primary human osteoblast/osteoclast co-cultures and seeded them on flat Ti and three Ti nanosurfaces with increasing degrees of roughness, manufactured using anodisation under alkaline conditions (for 2, 2.5 and 3 hours). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial growth on the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested, the 2 h nanowire surface supported osteoblast and, to a lesser extent, osteoclast growth and differentiation. Bacterial viability was significantly reduced on the 2h surface. Hence the 2 h surface provided optimal bone remodelling conditions while reducing infection risk, making it a favourable candidate for future implant surfaces. This work was funded by EPSRC grant EP/K034898/1.

Development of more effective diagnostic and therapeutic solutions is vital to tackling the growing challenge of bone diseases and disorders in aging societies. Spatially offset Raman spectroscopy (SORS) enables the chemical specificity of conventional Raman spectroscopy to be combined with sub-surface probing. SORS has successfully been applied to transcutaneous investigations of underlying bone and shows great potential to become an in vivo tool for non-invasive diagnosis of various bone conditions.

The volume within the complex hierarchical bone tissue probed by SORS depends on the specimen's optical properties. Understanding the actual sampling depth is important to correctly assign detected chemical changes to specific areas in the bone. This study explores the hypothesis that the effective Raman signal recovery from certain depths requires different spatial offsets depending on the bone mineralisation.

SORS depth investigations were conducted on three bones with significantly different mineralisation levels. Thin slices (0.6 – 1.0 mm thickness) were cut from deer antler, horse metacarpal and whale tympanic bulla and stacked together (4 – 7 layers; 4.1 – 4.7 mm total thickness). A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as reference sample and was inserted in between the layers of stacked bone slices. Raman spectra were acquired at 30 s using 830 nm excitation.

A quantitative relation between the SORS offset and the primarily interrogated depth inside the bone was established. Maximum accessible depths at small offset strongly depend on the mineralisation level. Using large spatial offsets of 7 – 9 mm PTFE signal recovery depths of 4.4 – 4.6 mm through cortical bone can be realized with only minor dependence on the bone mineralisation.

These findings highlight the potential of SORS for medical diagnostics by enabling the non-invasive detection of bone conditions characterised by chemical alterations several millimetres inside compact bone tissue (e.g. infections, tumours, etc.).

Osteoarthritis is one of the most common musculoskeletal diseases. It involves degeneration and loss of articular cartilage, leading to a painful bone on bone articulation during movement. Numerical FEA models exist to predict the mechanical behaviour of degenerated cartilage. One of the limitations of these models arises from the poor validation that can be attained with traditional experimental data. This typically relies on comparison with global mechanical quantities such as total tissue strain, which mask the individual contributions originating from the different layers. In order to improve on this, an experimental method was developed to visualise the through-thickness behaviour of articular cartilage.

Four experiments were performed on hemi-cylindrical cartilage plugs, harvested from a porcine femoral head, and immersed in a fluid solution. An Indian ink speckle pattern was applied to the flat surface of each hemi-cylinder. The specimens were equilibrated in 2.5M NaCl solution, transferred to a custom designed testing rig, and a reference image of the tissue cross-section was taken.

The solution concentration was then decreased to 0.15M and, predictably, the tissue thickness changed. Images of the tissue cross section were taken every 60s for the duration of the experiment (3600s). All images were analysed using a DIC algorithm (Ncorr open-source 2D digital image correlation matlab program), and documented the strain changes through the tissue thickness as a function of time. The measured total strain in the tissue was consistent with that reported by Lai et al. (1991). However the present technique allows to quantify the strain contribution from any of the tissue layers or sublayer. This poses a significant advantage over traditional methods as resulting information can further the understanding of the factors contributing to the mechanical behaviour of the tissue and provides an ideal platform for validating more and more refined models of tissue behaviour.

Edge loading due to dynamic separation can occur due to variations in component positioning such as a steep cup inclination angle (rotational) or mismatch between the centres of rotation of the head and the cup (translational). The aim of this study was to determine the effect of variations in rotational and translational positioning of the cup on the magnitude of dynamic separation, wear and deformation of metal-on-polyethylene bearings.

Eighteen 36mm diameter metal-on-polyethylene hip replacements were tested on an electromechanical hip simulator. Standard gait with concentric head and cup centres were applied for cups inclined at 45° (n=3) and 65° (n=3) for two million cycles. A further two tests with translational mismatch of 4mm applied between the head and cup bearing centres for cups inclined at 45° (n=6) and 65° (n=6) were run for three million cycles. Wear was determined using a microbalance and deformation by geometric analysis. Confidence intervals of 95% were calculated for mean values, and t-tests and ANOVA were used for statistical analysis (p<0.05).

Under 4mm mismatch conditions, a steeper cup inclination angle of 65° resulted in larger dynamic separation (2.1±0.5mm) compared with cups inclined at 45° (0.9±0.2mm). This resulted in larger penetration at the rim under 65° (0.28±0.04mm) compared to 45° (0.10±0.05mm) cup inclination conditions (p<0.01). Wear rates under standard concentric conditions were 12.8±3.8 mm³/million cycles and 15.4±5.0 mm³/million cycles for cups inclined at 45° and 65° respectively. Higher wear rates were observed under 4mm of translational mismatch compared with standard concentric conditions at 45° (21.5±5.5 mm³/million cycles, p<0.01) and 65° (23.0±5.7 mm³/million cycles, p<0.01) cup inclination.

Edge loading under dynamic separation conditions due to translational mismatch resulted in increased wear and deformation of the polyethylene liner. Minimising the occurrence and severity of edge loading through optimal component positioning may reduce the clinical failure rates of polyethylene.

Since the publication by Berger in 1993, many total knee replacements (TKR) have been measured using his technique to assess component rotation. Whereas the femoral landmarks have been showed to be accurate and precise, the use of the tibial tuberosity to ascertain the true tibial orientation is more controversial. The goal of this study was to identify a new anatomical landmark to measure tibial component rotation.

211 CTs performed after TKR were reviewed. The authors noticed that the lateral cortex of the tibia below the tibial plateau component was flat over a depth of approximately 10mm. A protocol to measure tibial rotation in relation to this landmark was developed: the slice below the tibial plateau was identified; a primary line was drawn over the straight lateral cortex of the tibia; a perpendicular to this line defined the reference axis (A); the posterior tibial component axis was drawn (B); the angle between A and B was measured with internal rotation being negative and external positive. Two independent observers measured 31 CTs twice each and Intraclass Correlation Coefficients (ICC) were calculated for intra- and inter-observer error. The 211CTs were measured according to Berger's and this protocol.

Intra-observer ICCs were 0.812 for Observer1 and 0.806 for Observer2. The inter-observer ICCs were 0.699 for Reading1 and 0.752 for Reading2. The Berger protocol mean tibial rotation was 9.7°±5.5° (−29.0° to 5.2°) and for the new landmark 0°±5.4° (−18.6° to 14°).

This new tibial landmark appeared easy to identify and intra- and inter-observer errors were acceptable. The fact that the mean tibial rotation was 0° makes this landmark attractive. A consistent easily identified landmark for tibial rotation may allow for improvement in component rotation and the diagnosis of dissatisfaction after TKR. Further studies are under way to confirm the relevance of this landmark.

Patients with osteoarthritis (OA) of the knee commonly alter their movement to compensate for deficiencies. This study presents a new numerical procedure for classifying sit-to-walk (STW) movement strategies.

Ten control and twelve OA participants performed the STW task in a motion capture laboratory. A full body biomechanical model was used. Participants were instructed to sit in a comfortable self-selected position on a stool height adjusted to 100% of their knee height and then stand and pick up an object from a table in front of them. Three matrices were constructed defining the progression of the torso, feet and hands in the sagittal plane along with a fourth expressing the location of the hands relative to the knees. Hierarchical clustering (HC) was used to identify different strategies. Trials were also classified as to whether the left (L) and right (R) extremities used a matching strategy (bilateral) or not (asymmetrical). Fisher's exact test was used to compare this between groups.

Clustering of the torso matrix dichotomised the trials in two major clusters; subjects leaning forward (LF) or not. The feet and hands matrices revealed sliding the foot backward (FB) and moving an arm forward (AF) strategies respectively. Trials not belonging in the AF cluster were submitted to the last HC of the fourth matrix exposing three additional strategies, the arm pushing through chair (PC), arm pushing through knee (PK) and arm not used (NA). The control participants used the LF+FBR+PK combination most frequently whereas the OA participants used the AFR+PCL. OA patients used significantly more asymmetrical arm strategies, p=0.034.

The results demonstrated that control and OA participants favour different STW strategies. The OA patients asymmetrical arm behaviour possibly indicates compensating for weakness of the affected leg. These strategy definitions may be useful to assess post-operative outcomes and rehabilitation progress.

Patellofemoral pain syndrome (PFPS) is a common knee disorder in active individuals. Movement dysfunction of valgus positioning at the knee during weight-bearing is frequently seen in PFPS. A single-leg squat (SLS) is a test commonly used in physiotherapy to assess for movement dysfunction. Kinesio-Tape (KT) is gaining in popularity in treating PFPS and claims to alter muscle recruitment and motor control, however evidence is weak. Objective: To evaluate the effect of KT applied to the quadriceps on muscle activity with electromyography (EMG) of the rectus femoris, vastus lateralis and vastus medialis oblique and motor control via the frontal plane projection angle (FPPA) using 2-dimensional video analysis.

A convenience sample of healthy females were recruited and performed 5 single-leg squats with and without KT. EMG of the quadriceps was recorded and dynamic valgus assessed via the FPPA using Dartfish video analysis software. Eccentric and concentric EMG data was recorded and the FPPA measured in single-leg stance and the depth of the squat. Institutional ethical approval was obtained for the study.

16 active females were assessed (mean age 28.94 +6.58 years). Wilcoxon signed-rank tests found no significant change in eccentric or concentric EMG of the quadriceps (%MVC) with KT compared to without (p values 0.35–0.86). Paired-sample t-tests found no significant difference in FPPA between conditions in single-leg stance (p=1.00) or the depth of the squat (p=0.871).

KT did not affect EMG activity of the quadriceps or the FPPA in a SLS when applied to the quadriceps of healthy females, questioning proposed effects of KT on normal muscle tissue. Further research is required into the efficacy of using KT in physiotherapy.

The increase in revision joint replacement surgery and fractures of bone around orthopaedic implants may be partly addressed by keeping bone healthy around orthopaedic implants by inserting implants with mechanical properties closer to the patient's bone properties. We do not currently have an accurate way of calculating a patient's bone mechanical properties. We are therefore investigating whether microindentation can accurately calculate bone stiffness.

We received ethical approval to retrieve femoral heads and necks from patients undergoing hip replacement surgery for research. Cortical bone from the medial calcar region of the femoral neck was cut into 3×3×6mm cuboid specimens. Micro-indentation testing was performed in the direction of loading of the bone using a MicroMaterials indenter. The samples were kept hydrated and were not fixed or polished. From the unloading curve after indentation, the elastic modulus was calculated, using the Oliver- Pharr method. To assess which microindentation machine settings most precisely calculate the elastic modulus we varied the loading and unloading rates, load and indenter tip shape.

The most precise results were obtained by using a spherical indenter tip (rather than Berkovich tip), high load (10N), a loading rate of 100 mN/s and unloading rate of 300 mN/s with a pause of 60 seconds at maximum load and multiple load cycles with constant loads. Using these settings the mean elastic modulus over 12 cycles of testing was 13.0 GPa (+/- 2.47).

By using a spherical indenter tip and fast unloading it was possible to get precise apparent modulus values. By unloading as fast as possible the effects of bone viscoelastic properties are minimised. By using a spherical indenter tip, plastic deformation at the tip is minimised (compared to the Berkovich tip). We are performing further standard compression tests on the samples to verify the accuracy of the indentation tests.

Physical outcome following total knee arthroplasty is variable. Satellite cells are undifferentiated myogenic precursors considered to be muscle stem cells. We hypothesised that; the recovery of muscle strength and physical function following knee arthroplasty would be influenced by the underlying number of muscle satellite cells.

16 patients provided a distal quadriceps muscle biopsy at time of surgery. Satellite cells were identified with a primary mouse antibody for Pax7 – a cytoplasmic protein marker, and the myonuclei with DAPI. Positive cells were identified on the basis of immunofluorescent staining in association with nuclear material, and confirmed by position under the basal lamina. Patient function was assessed using a validated physical assessment protocol, the Aggregated Locomotor Function (ALF) score, muscle strength assessed using the leg extensor power-rig, and clinical outcome assessed with the Oxford Knee Score (OKS) pre-operatively and at 1 year post operatively.

Muscle satellite cell content varied amongst the patient group (Positive Staining Index 3.1 to 11.4). Satellite cell content at time of surgery correlated with change in outcomes between pre-operative and 1 year assessments in all assessed parameters (ALF, r = 0.31; muscle power, r = 49; OKS, r = 0.33). Regression analysis employing a forward stepwise selection technique employed satellite cell volume in models of pre-operative to 1 year change for all outcome parameters. Physical function (satellite cell content, patient age and pre-operative ALF score) adjusted R2 = 0.92; Muscle power (pre-operative power and satellite cell content) adjusted R2 = 0.38; Clinical outcome (pre-operative OKS and satellite cell content) adjusted R2 = 0.28.

Muscle satellite cell content influences recovery of muscle power and physical function following total knee arthroplasty. Importantly it is also associated with change in clinical scores; suggesting it to be a biomarker for patient outcomes.

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice.

Our aim is to investigate the role of TRIM32 in human and murine articular tissue.

TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation.

TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10⁻⁵), and Sox9 (p=2.35×10⁻⁶). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006).

These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation.

Systemic concentrations of metal ions (cobalt and chromium) are persistently elevated in patients with metal-on-metal hip resurfacing (MOMHR) compared to conventional total hip arthroplasty (THA). Several studies by us and others have described the detrimental effects of metal exposure on survival and function of various cell types in-vitro, but the mechanisms for these effects remain unclear. Epigenetic modifications following chronic metal exposure is a possible mechanism that could mediate these effects. Here we test the methylation status in genomic DNA from MOMHR (“cases”) and THA (“controls”) patient-groups, and its correlation with circulating metal levels.

The cohort consisted of 34 patients with a well-functioning MOMHR at a median follow-up of 9.75 years. These were individually matched for gender, age and time-since-surgery to a non-exposure group consisting of patients with THA. Genomic DNA was isolated from blood samples and cell composition estimated using the ‘estimateCellCounts’ function in ‘minfi R-package’. Methylation was assessed using the Illumina 450k BeadChip array analysing 426,225 probes. Logit model was fitted at each probe with case/control status as independent variable and covariates of gender, age, time-since-surgery, smoking, non-arthroplasty metal exposure, and cell composition. DNA methylation age was assessed using an online calculator (https://dnamage.genetics.ucla.edu/) and comparisons made between cases and controls, and correlated with circulating metal levels.

Cell distributions did not differ between the cases and controls (Wilcoxon test p<0.17) with no probe having an association at 5% FDR. Circulating metal levels and LVEDD also had no association with any probe at 5% FDR. There was no preferential age acceleration between cases and controls (Wilcox p<0.7), and it had no correlation with plasma-chromium or blood-cobalt levels (p<0.9).

In summary, large methylation changes following MOMHR seem to be absent, compared to THA. Future research with larger samples will be needed to clarify the presence and extent of small methylation changes.

Wear particles produced by alumina ceramic-on-ceramic (CoC) bearings cause a minimal immunological response with low cytotoxicity and inflammatory potential^{1, 2}. However, more comprehensive immunological studies are yet to be completed for the composite CoC (zirconia-toughened, platelet reinforced alumina) hip replacements due to difficulties in isolating the very low volume of clinically relevant wear debris generated by such materials in vitro. The aim of this study was to compare the cytotoxic effects of clinically relevant cobalt chromium (CoCr) nano-particles with commercial composite ceramic particles.

Composite ceramic particles (commercial BIOLOX® delta powder) were obtained from CeramTec, Germany and clinically relevant CoCr wear particles were generated using a six station pin-on-plate wear simulator. L929 fibroblast cells were cultured with 50µm³ of CoCr wear debris or composite ceramic particles at low to high volumes ranging from 500µm³–0.5µm³ per cell and the cyctotoxic effects of the particles were assessed over a period of 6 days using the ATP-Lite™ cell viability assay.

The composite ceramic particles were bimodal in size (0.1–2µm & 30–100nm) and showed mild cytotoxic effects when compared with equivalent particle volumes (50µm³) of clinically relevant CoCr nano-particles (10–120nm). The CoCr nano-particles had significant cytotoxic effects from day 1, whereas the composite ceramic particles only showed cytotoxic effects at particle concentrations of 50 and 500µm³ after 6 days. The increased cytotoxicity of the clinically relevant CoCr nano-particles may have been attributed to the release of Co and Cr ions.

This study demonstrated the potential cytotoxic effects of model ceramic particles at very high volume concentrations, but it is unlikely that such high particle volumes will be experienced routinely in vivo in such low wearing bearing materials. Future work will investigate the longer-term effects on genotoxicity and oxidative stress of low volumes of clinically-relevant generated BIOLOX® delta ceramic wear particles.

We have previously observed an increase in total bone mineral density and reduced bone turnover (TRAP5b and osteocalcin) in patients with well-functioning metal-on-metal hip resurfacing (MOMHR). Here, we provide data to support the hypothesis that osteoclast differentiation and function is altered in this patient population, and that this effect is transferrable through their serum.

Patients with well-functioning MOMHR (cases, n=18) at a median follow-up of 8 years were individually matched for gender, age and time-since-surgery to a low-exposure group consisting of patients with THA (controls, n=18). The monocyte fraction of patient peripheral blood was isolated and differentiated into osteoclasts on dentine wafers using RANKL and M-CSF supplemented media (osteoclastogenic media, OM). Cultures were monitored for the onset of resorption, at which point the cells were treated with OM, autologous serum or serum from matched MOMHR/THA donors, all supplemented with RANKL and M-CSF. At the end of the culture, cells were TRAP-stained and quantified using CellD Software Package, Olympus.

When cells were differentiated in standard osteoclastogenic media, the resorbing ability of osteoclasts derived from MOMHR patients was reduced 22%(p<0.0079) compared to THA. The resorbing ability of osteoclasts generated from MOMHR patients and differentiated in autologous serum was reduced 33%(p<0.0001), whilst matched THA serum caused a smaller reduction of 14%(p<0.01). When cells derived from THA patients were differentiated in autologous serum, the resorbing ability of osteoclasts was similarly reduced by 35%(p<0.0001), whilst the matched MOMHR serum also caused a reduction of 21%(p<0.0001).

This data suggests that prior exposure to higher circulating Co and Cr in patients with MOMHR reduces osteoclastogenesis, and that the detrimental effect on the functionality of mature osteoclasts is transferable through the serum. This has implications for systemic bone health of patients with MOMHR or modular taper junctions.

A subgroup of patients that undergo TKR surgery have evidence of neuropathic pain and central sensitization that may predispose to severe postoperative pain. This study assesses the correlation of MRI detected bone marrow lesions (BMLs) and synovitis with markers of neuropathic pain and central sensitization in patients undergoing TKR surgery and healthy volunteers.

31 patients awaiting TKR and 5 healthy volunteers were recruited. Each subject underwent a 3-T knee MRI scan that was graded for BMLs (0–45) and synovitis (0–3) using subsets of the MRI Osteoarthritis Knee Score (MOAKS). All subjects were asked to complete the PainDetect questionnaire to identify nociceptive pain (< 13), unclear pain (13–18) and neuropathic pain (>18). Correlation between BMLs and PainDetect score was the primary outcome measure. Secondary outcomes included the correlation of synovitis to PainDetect and temporal summation (TS) a measure of central sensitization to the PainDetect score. TS was determined using a monofilament to evoke pain. Pilot histological analysis of the prevalence of osteoclasts (TRAP⁺) within BMLs versus normal subchondral bone was performed, implying a role in BML pathology.

Increasing BML MOAKS score correlated with neuropathic pain (painDetect), r_s = 0.38, p=0.013 (one-tailed). There was a positive correlation between synovitis and PainDetect score, τ =0.23, p= 0.031 (one-tailed). TS was greater in the neuropathic pain than in nociceptive pain patients, U = 18.0, p=0.003 (one-tailed). TRAP staining identified more osteoclasts within BMLs than contralateral condyle lesion free subchondral bone, z = −2.232, p = 0.026 (Wilcoxon Signed Rank Test, one-tailed).

BMLs and synovitis are more prevalent in neuropathic pain and central sensitization in knee OA. Higher osteoclast prevalence was seen within BMLs which may help explain the association with BMLs and pain in OA.

Femoral head collapse due to avascular necrosis (AVN) is a relatively rare occurrence following intertrochanteric fractures; however, with over thirty-thousand intertrochanteric fractures per year in England and Wales alone, and an incidence of up to 1.16%, it is still significant. Often patients are treated with a hip fixation device, such as a sliding hip screw or X-Bolt. This study aimed to investigate the influence of three factors on the likelihood of head collapse: (1) implant type; (2) the size of the femoral head; and (3) the size of the AVN lesion.

Finite element (FE) models of an intact femur, and femurs implanted with two common hip fixation designs, the Compression Hip Screw (Smith & Nephew) and the X-Bolt (X-Bolt Orthopaedics), were developed. Experimental validation of the FE models on 4^th generation Sawbones composite femurs (n=5) found the peak failure loads predicted by the implanted model was accurate to within 14%. Following validation on Sawbones, the material modulus (E) was updated to represent cancellous (E=500MPa) and cortical (E=1GPa) bone, and the influence of implant design, head size, and AVN was examined. Four head sizes were compared: mean male (48.4 mm) and female (42.2 mm) head sizes ± two standard deviations. A conical representation of an AVN lesion with a lower modulus (1MPa) was created, and four different radii were studied. The risk of head collapse was assessed from (1) the critical buckling pressure and (2) the peak failure stress.

The likelihood of head collapse was reduced by implantation of either fixation device. Smaller head sizes and greater AVN lesion size increased the risk of femoral head collapse. These results indicate the treatment of intertrochanteric fractures with a hip fixation device does not increase the risk of head collapse; however, patient factors such as small head size and AVN severity significantly increase the risk.

Painful OA is linked to CNS changes in pain processing. Temporal summation of pain (TSP) is a measure of one such CNS change, central sensitization. TSP is defined using a series (≥0.33Hz) of painful stimuli and is a predictor of postoperative pain, experienced by 20% of patients after total knee replacement (TKR) surgery. This study has developed a protocol to use functional MRI to assess CNS changes in OA pain processing.

This pilot includes 3 participants with chronic knee OA pain awaiting TKR (62 ± 4.4) and 5 healthy volunteers (50 ± 13.6). 3-Tesla BOLD fMRI brain scans were recorded during short series of one second painful stimuli, applied using an automated inflatable cuff to the calf muscle of the leg with the affected knee or left side in healthy volunteers. The pain intensity at onset and during the 10 painful stimuli were recorded using a numerical rating scale. The pattern of brain activation was averaged across noxious stimuli, and the differential activation compared the 1st vs. 10th (last) stimulus. Bone marrow lesions (BMLs), synovitis and effusion size were scored from 3-Tesla knee MRI's using MOAKS scoring.

TSP was raised in OA patients compared to control group (p=0.023). TSP brain activity in the chronic OA patients displayed higher signal within the subgenual anterior cingulate (sgACC) compared to healthy volunteers. Knee MRI identified OA patient's exhibited higher BML scores (p=0.038) and more knee effusion (p=0.018), but the lack of synovitis did not differ from control group (p=0.107).

Enhanced TSP in chronic knee OA pain may be linked with augmented responses in emotional circuitry. BMLs and effusion size appear to contribute more with pain than synovitis. These results may help understand sensitization to improve outcomes for patients with knee OA undergoing TKR surgery.

Patient function is poorly characterised following revision TKA. Modern semi-constrained implants are suggested to offer high levels of function, however, data is lacking to justify this claim.

52 consecutive aseptic revision TKA procedures performed at a single centre were prospectively evaluated; all were revision of a primary implant to a Triathlon total stabiliser prosthesis. Patients were assessed pre-operatively and at 6, 26, 52 and 104 weeks post-op. Outcome assessments were the Oxford Knee Score (OKS), range of motion, pain rating scale and timed functional assessment battery. Analysis was by repeated measures ANOVA with post-hoc Tukey HSD 95% simultaneous confidence intervals as pairwise comparison. Secondary analysis compared the results of this revision cohort to previously reported primary TKA data, performed by the same surgeons, with identical outcome assessments at equivalent time points.

Mean age was 73.23 (SD 10.41) years, 57% were male. Mean time since index surgery was 9.03 (SD 5.6) years. 3 patients were lost to follow-up. All outcome parameters improved significantly over time (p <0.001). Post-hoc analysis demonstrated that all outcomes changed between pre-op, 6 week and 26 weeks post-op assessments.

No difference was seen between primary and revision cohorts in OKS (p = 0.2) or pain scores (p=0.19). Range of motion and functional performance was different between groups over the 2 year period (p=0.03), however this was due to differing pre-operative scores, post-hoc analysis showed no difference between groups at any post-operative time point.

Patients undergoing aseptic revision TKA with semi-constrained implants made substantial improvements in OKS, pain scores, knee flexion, and timed functional performance, with the outcomes achieved comparable to those of primary TKA. High levels of function can be achieved following revision knee arthroplasty, which may be important considering the changing need for, and demographics of, revision surgery.

Edge loading due to dynamic separation can occur due to variations in component positioning such as a steep cup inclination angle (rotational) or mismatch between the centres of rotation of the head and the cup (translational). The aim of this study was to determine the effect of variations in rotational and translational positioning of the cup on the magnitude of dynamic separation, wear and deformation of metal-on-polyethylene bearings.

Eighteen 36mm diameter metal-on-polyethylene hip replacements were tested on an electromechanical hip simulator. Standard gait with concentric head and cup centres were applied for cups inclined at 45° (n=3) and 65° (n=3) for two million cycles. A further two tests with translational mismatch of 4mm applied between the head and cup bearing centres for cups inclined at 45° (n=6) and 65° (n=6) were run for three million cycles. Wear was determined using a microbalance and deformation by geometric analysis. Confidence intervals of 95% were calculated for mean values, and t-tests and ANOVA were used for statistical analysis (p<0.05).

Under 4mm mismatch conditions, a steeper cup inclination angle of 65° resulted in larger dynamic separation (2.1±0.5mm) compared with cups inclined at 45° (0.9±0.2mm). This resulted in larger penetration at the rim under 65° (0.28±0.04mm) compared to 45° (0.10±0.09mm) cup inclination conditions (p<0.01). Wear rates under standard concentric conditions were 12.8±3.8 mm³/million cycles and 15.4±5.0 mm³/million cycles for cups inclined at 45° and 65° respectively. Higher wear rates were observed under 4mm of translational mismatch compared with standard concentric conditions at 45° (21.5±5.5 mm³/million cycles, p<0.01) and 65° (23.0±5.7 mm³/million cycles, p<0.01) cup inclination.

Edge loading under dynamic separation conditions due to translational mismatch resulted in increased wear and deformation of the polyethylene liner. Minimising the occurrence and severity of edge loading through optimal component positioning may reduce the clinical failure rates of polyethylene.

Surgical interventions for the treatment of chronic neck pain, which affects 330 million people globally [1], include fusion and cervical total disc replacement (CTDR). Most of the currently clinically available CTDRs designs include a metal-on-polymer (MoP) bearing. Numerous studies suggest that MoP CTDRs are associated with issues similar to those affecting other MoP joint replacement devices, including excessive wear and wear particle-related inflammation and osteolysis [2,3]. A device with a metal-on-metal (MoM) bearing has been investigated in the current study.

Six MoM CTDRs made from high carbon cobalt-chromium (CoCr) were tested in a six-axis spine simulator, under standard ISO testing protocol (ISO-18192-1) for a duration of 4 million cycles (MC). Foetal bovine calf serum (25%v/v), used as a lubricant, was changed every 3.3×10⁵ cycles and saved for particle analysis. Components were taken down for measurements after each 10⁶ cycles; surface roughness, damage modes and gravimetric wear were assessed.

The mean wear rate of the MoM CTDRs was 0.24mm³/MC (SD=0.03), with the total volume of 0.98mm³ (SD=0.01) lost over the test duration. Throughout the test, the volumetric wear was linear; no significant bedding-in period was observed. The mean pre-test surface roughness decreased from 0.019μm (SD=0.005) to 0.012μm (SD=0.002) after 4MC of testing. Prior to testing, fine polishing marks on the bearing surfaces were observed using light microscopy. Following 4MC of testing, these polishing marks had been removed. Consistently across all components, surface discolouration and multidirectional, criss-crossing, circular wear tracks, caused by abrasive wear, were observed.

The wear results showed low wear rates exhibited by MoM CTDRs (0.24mm³/MC), when compared CTDR designs incorporating metal-on-polymer bearings (0.56mm³/MC) [4] as well as MoM lumbar CTDRs [5,6] (0.76mm3/MC – 6.2mm³/MC). These findings suggest that MoM CTDRs are more wear resistant than MoP CTDRs, however the particle characterisation and biological consequences of wear remain to be determined.

Obese patients undergoing total knee arthroplasty (TKA) face increased risks of complications such as joint infection and early revision. However, the influence of obesity on measures of patient function following TKA is poorly defined.

Knee arthroplasty outcome data for procedures carried out over an eight month period was extracted from a regional database in the UK. We analysed the impact of weight categories (BMI<30, BMI=30–34.9, and BMI≥35) on the Forgotten Joint Score – 12 (FJS-12) and Oxford Knee Score (OKS). Data was available preoperatively and 12 months postoperatively. Physical and mental health was assessed with the SF-12 one year after surgery.

Data from 256 patients were available. 49.6% had a BMI<30, 27.4% had a BMI 30–34.9 and 23.1% had a BMI≥35. Mean FJS-12 results at 1-year were 48.7 points for patients with a BMI<30, 40.7 points for patients with a BMI=30–34.9 and 34.0 points for patients with a BMI≥35. Effect sizes for change from baseline to 12-month post-op were 3.0 (Cohen's d) in patients with BMI<30 and d=2.2 in patients with BMI≥35. Mean OKS results at 1 year were 36.9 (BMI<30), 33.7 (BMI=30–34.9) and 32.0 (BMI≥35) respectively. Effect sizes for change from baseline to 12-month was d=2.1 (BMI<30) and d=1.9 (BMI≥35). Differences between BMI groups with regard to post-operative change were statistically significant for the FJS-12 (p=0.038) but not for the OKS (p=0.229).

This study highlights that outcome scores may differ in their ability to capture the impact of obesity on patient function following TKA. The FJS-12 showed significant differences in outcome based on patient obesity category, whereas the OKS did not detect between group differences.

Total ankle replacement (TAR) has a mean survivorship of 77% at 10 years which is poor compared to other types of joint arthroplasty. Osteolysis and aseptic loosening are commonly cited TAR failure modes, the mechanisms of which are unknown. Retrieval analyses of TAR devices may reveal mechanisms of failure similar or dissimilar to other joint replacements. This study investigated whether TAR explants exhibit similar damage modes to those recognised in other total joint replacements.

22 Ankle Evolution System TARs (Transystème, Nimes, France) were implanted and retrieved by the same surgeon. Mean implantation time was 7.8 yrs (5.3 to 12.1 range). Pain and/or loosening were the indications for revision. Macro photography, an Alicona Infinite microscope and the Hood/Wasielewski scale were used to classify damage modes on the polyethylene insert. Scanning electron microscopy with energy dispersive X-ray spectroscopy was used to determine the composition of third body debris and to image the fixation surface of the tibial components.

Mean damage score was 185.4 (± 40.0 SD). Damage modes common to total knee replacements were identified on both the superior and inferior insert surfaces, these included: burnishing, scratching, pitting and abrasion. Titanium particles, hydroxyapatite fragments and bone debris were embedded in the insert surfaces. Fixation surface delamination was identified by the ongrowth of tissue between the cobalt chromium substrate and titanium alloy coating.

Damage modes indicative of high levels of wear and deformation were evident. Pitting caused by third body debris was abundant and suggested fixation surface wear and failure.

Osteoarthritis (OA) affects bone cartilage and underlying bone. Mechanically, the underlying bone provides support to the healthy growth of the overlying cartilage. However, with the progress of OA, bone losses and cysts occur in the bone and these would alter the biomechanical behaviour of the joint, and further leading to bone remodelling adversely affect the overlying cartilage.

Human femoral head and femoral condyle were collected during hip or knee replacement operation due to the end stage of osteoarthritis (age 50–70), and the cartilage patches were graded and marked. A volunteer patient, with minor cartilage injury in his left knee while the right knee is intact, was used as control. Peripheral quantitative computed tomography (pQCT) was used to scan the bone and to determine the volumetric bone mineral density (vBMD) distribution.

The examination of retrieved tissue explants from osteoarthritic patients revealed that patches of cartilage were worn away from the articular surface, and patches of intact cartilage were left. The cysts, ranging from 1 to 10mm were existed in all osteoarthritic bones, and were located close to cartilage defects in the weight-bearing regions, and closely associated with the grade of cartilage defect as measured by pQCT. The bone mineral density (vBMD) distribution demonstrated that the bones around cysts had much higher vBMD than the trabecular bone away from the cysts. Compared to the subchondral bone under thicker cartilage, subchondral bone within cartilage defect has higher vBMD. This may result from the mechanical stimulation as a result of bone-bone direct contact with less protection of cartilage in cartilage defect regions.

This study showed an association between cartilage defect and subchondral bone mineral density distribution. Cysts were observed in all osteoarthritic samples and they are located close to cartilage defects in the weight-bearing regions. Cartilage defect altered the loading pattern of the joints, this leading to the bone remodelling and resultant bone structural changes as compared to the normal bone tissues.

This work was financially supported by The ARUK Proof of Concept Award (grant no: 21160).

Adductor canal blocks offer an alternative to femoral nerve block for postoperative pain relief in knee arthroplasty. They may reduce the risk of quadriceps weakness, allowing earlier mobilisation of patients postoperatively. However, little is known about the effect of a tourniquet on the distribution of local anaesthetic in the limb.

Ultrasound-guided adductor canal blocks were performed on both thighs of five human cadavers. Left and right thighs of each cadaver were randomised to tourniquet or no tourniquet for one hour. Iohexol radio-opaque contrast (Omnipaque 350) was substituted for the local anaesthetic for X-Ray imaging. All limbs underwent periodic flexion and extension during this hour to simulate positioning during surgery. The cadavers were refrozen. Fiducial markers were inserted into the frozen tissue. X-rays were obtained in 4 planes (AP, lateral 45° oblique/medial oblique, lateral). University Research Ethics Approval was obtained and cadavers were all pre-consented for research, imaging and photography according to the Anatomy Act (1984).

Analysis of radiographs showed contrast distribution in all thighs to be predominantly on the medial aspect of the thighs. The contrast margins were entire and well circumscribed, strongly suggesting it was largely contained within the aponeurosis of the adductor canal. Tourniquets appeared to push the contrast into a narrower and more distal spread along the length of the thigh compared to a more diffuse spread for those without. Proximal spread towards the femoral triangle was reduced in limbs without tourniquets.

The results suggest that contrast material may remain within the adductor canal structures during adductor canal blocks. Tourniquets may cause greater distribution of contrast proximally and distally in the thigh, but this does not appear to be clinically significant. Further studies might include radio-stereo photometric analysis using the fiducial markers in the limbs and in vivo studies to show the effect of haemodynamics on distribution.

Three distal femoral axes have been described to aid in alignment of the femoral component; the Trans Epicondylar Axis (TEA), the Posterior Condylar Axis (PCA) and the Antero Posterior (AP) axis. Our aim was to identify if there was a reproducible relationship between the axes which would aid alignment of the femoral component. This is the first study compare all three distal femoral axes with each other using magnetic resonance imaging (MRI) in a Caucasian population. Our sample group represents real life patients awaiting total knee arthroplasty (TKA), as opposed non-arthritic or cadaveric knees.

We identified the relationship between these rotational axes by performing MRI scans on 89 patients awaiting TKA with patient-specific instrumentation. Measurements were taken by two observers.

Patients had a mean age of 62.5 years (range 32–91). 51 patients were female. The mean angle between the TEA and the AP axis was 92.78° with a standard deviation of 2.51° (range 88° – 99°). The mean angle between the AP axis and the PCA was 95.43° with a standard deviation of 2.75° (range 85° – 105°). The mean angle between the TEA and the PCA was 2.78° with a standard deviation of 1.91° (range 0° – 10°).

We conclude that while there is a reproducible relationship between the differing femoral axes, there is a significant range in the relationship between the femoral axes. This range may lead to greater inaccuracy than has previously been appreciated when defining the rotation of the femoral component. There is most variation between the PCA and the AP axis. The TEA's relationship with the PCA and AP appears important in defining rotation. Due to the well accepted difficulty in defining the TEA intra-operatively, there may be a role for patient-specific instrumentation in TKA surgery with pre-operative MRI.

The pathogenesis of falling bone mineral density (BMD) as a universal feature of advancing age is poorly understood¹. Frequently culminating in the development of osteoporosis, the process is attributable to more than 500,000 fragility fractures occurring every year in the UK Such injuries are associated with great levels of morbidity, mortality and a £3.5 billion cost to the healthcare economy².

With age, humans are known to accumulate somatic mitochondrial DNA (mtDNA) mutations in mitotic and post mitotic tissue, and stem cell precursors³. Compelling evidence in recent years, particularly that provided by animal models suggests that these mutations are intrinsic to the ageing process^4–6. We provide evidence for the first time that mitochondrial dysfunction contributes significantly to the failure of bone homeostasis and falling BMD.

We have utilised a mouse model that accumulates mtDNA mutations at 3–5 times the rate of normal mice, consequently ageing and developing osteoporosis prematurely⁷, to clearly demonstrate that osteoblasts are vulnerable to mtDNA mutations. We have developed a new quadruple immunofluorescent assay to show that mitochondrial respiratory chain dysfunction occurs in osteoblasts as a consequence (p < 0.0001). We show that this mitochondrial dysfunction is associated with reduced BMD in female and male mice by 7 (p = 0.003) and 11 (p = 0.0003) months of age respectively. Using osteoblasts derived from mesenchymal stem cells extracted from male and female mice with mitochondrial dysfunction aged 4, 7 and 11 months, we demonstrate a vastly reduced capacity to produce new mineralised bone in vitro when compared to wild type cell lines (p < 0.0001). Exercise was found to have no beneficial effect on osteoblast and whole bone phenotype in this mouse model. It is likely that mtDNA mutations accumulating over a longer time period in human ageing have significantly detrimental effects on bone biology and diminishing BMD.

There is increasing interest in using anabolic factors such as stem cells to augment fragility fracture repair. One of the factors associated with fracture healing is the retention and migration of stem cells to the site of injury (1–3). The aim of this study was to isolate stem cells from osteopenic rats and investigate and compare the CD marker expression, proliferation, migration, osteogenic and adipogenic differentiation. The hypothesis of this study is that the migration of MSCs from young, adult and ovariectomised (OVX) rats will have different proliferation, differentiation and migratory abilities.

CD marker expression of MSCs from young, adult and osteopenic rats was measured using flow cytometry. Proliferation, osteogenic differentiation and adipogenic differentiation was measured using Alamar Blue, ALP expression and Alizari n Red and quantitative Oil red O respectively. Cells were incubated in Boyden chambers to quantify their migration towards SDF1. Data was analysed using a Student t-test where p values < 0.05 were considered significant.

MSCs from all 3 groups of rats had similar proliferation and expression of CD29(>90%), CD90(>96%), CD34(<5%) and CD45(approx 10%). The proliferation rate was also similar. However, interestingly the migration and differentiation ability was significantly different between the MSCs from the 3 groups of rats. The young MSCs were not only better at differentiating into bone and fat, but they also migrated significantly more towards SDF1. MSCs from OVX rats are similar to MSCs from young rats. However when induced to turn into bone, fat and migrate towards SDF1, young MSCs are significantly more responsive than MSCs from OVX and adult control rats. The poor homing ability and differentiation of the stem cells and their retention may result in a reduction in bone formation leading to delayed union in fractures of osteoporotic patients(4).

Intermittent parathyroid hormone (iPTH 1–34) increases bone formation via modelling and remodelling mechanisms and as such is used to treat osteoporosis. The actions of iPTH on mesenchymal stem cell (MSCs) may underpin a further treatment option.

We isolated bone marrow derived MSCs from young (WT) and ovarectomized senile (OVX) rats, investigating the effect of intermittent and continuous PTH administration on migration to SDF-1, proliferation and osteogenic differentiation.

MSCs were harvested from the femora of 6–10week old WT rats and 10–13month old OVX rats. Cells were cultured with 25,50 and 100nmMol of PTH 1–34 added to osteogenic media either continuously or intermittently for 6hours in every 72hour cycle. ALP and Alizarin Red assessed osteogenic differentiation, and Alamar Blue- proliferation. Cells were seeded in a Boyden chamber to quantify SDF-1 migration. A student t-test was used to analyse results, and a p value<0.05 considered significant.

ALP and Alizarin Red were significantly increased for WT and OVX groups at 50nmMol of iPTH. Continuous administration at all concentrations reduced calcium phosphate deposition by day 21 in all groups.

In comparison to cells cultured in osteogenic media, 50nmMol of iPTH led to significantly higher ALP and Alizarin Red measurements up to days 10 and 7 respectively (figure 1). There was no change in proliferation between the groups, and PTH had no effect (figure 2.)

WT MSCs not only had improved osteogenic differentiation, but also showed increased migration to SDF-1 in comparison to OVX groups. iPTH led to further increases in migration of both OVX and WT cells.

iPTH increases the osteogenic differentiation and migration of MSCs from both young and ovarectomised rats, though this effect is not dose dependent. Ultimately, the role of iPTH on MSCs may lead to improved bone formation and cell homing capacity-particularly in the context of osteoporosis.

Bone has a number of different functions in the skeleton including the physical roles of support, protection and sound wave conduction. The mechanical properties, required for these different functions varies and can be achieved by compositional adaption of the bone material, in addition to changes in shape and architecture. A number of previous studies have demonstrated the relationship between mechanical function and mineral to collagen ratio in bones from different species.

The aim of this study is to test the hypothesis that the mineral to collagen ratio is higher in bone with a mechanically harder matrix within a species.

The red deer (Cervus elaphus) (n=6) was chosen as a model for studying bone with extreme properties. The mechanical properties of the antler, metacarpal bone and tympanic bulla were defined by indentation using a bench-top indentation platform (Biodent). The mineral to collagen ratio was quantified using Raman spectroscopy. The deposition of mineral was studied at macro-level using pQCT.

The results showed that the hardness (Indentation Distance Increase) was lowest in the metacarpal (8.5µm), followed by the bulla bone (9.4µm) and highest in the antler (14.5µm). Raman spectroscopy showed a mineral:collagen ratio of 1:0.10 (bulla), 1:0.13 (metacarpal) and 1:0.15 (antler) for the different bones. This does not follow the more linear trend previously shown between young's modulus and the mineral:collagen ratio. The location of the mineral appeared to differ between bone types with pQCT revealing locations of concentrated density and banding patterns in antler. Interestingly, Raman spectra showed differences in the amide peaks revealing differences in protein structure.

The results reject the hypothesis but also suggest that the organisation of mineral and collagen has an impact on the hardness modulus. We demonstrate that the red deer provides a good model for studying bone specialisation. This work will provide the basis for further investigation into collagen as a controlling factor in mineral deposition.

Osteoporosis is an international health and financial burden of ever increasing proportions. Current treatments limit the rate of bone resorption and reduce fracture risk, however they are often associated with significant and debilitating side effects. The most commonly used therapies also do not stimulate osteoblast activity. Much current research focus is aimed at the metabolic and epigenetic pathways involved in osteoporosis. MicroRNAs have been shown to play an important role in bone homeostasis and pathophysiological conditions of the musculoskeletal system. Upregulation of specific microRNAs has been identified in-vivo in osteoporotic patients. It is hypothesized that modulation of specific mircoRNA expression may have a key role in future targeted therapies of musculoskeletal diseases. The assessment and analysis of their potential therapeutic use in Osteoporosis is of great importance, due to the burden of the disease.

We have developed a 3D osteoporotic model from human bone marrow, without the use of scaffold. Magnetic nanoparticles are utilised to form spheroids, which provides a closer representation of the in-vivo environment than monolayer culture. This model will provide the basis for analysing future microRNA experiments to assess the potential upregulation of osteoblastogenesis without cessation of osteoclast activity.

The results of initial monolayer and spheroid experiments will be presented. Optimisation of the osteoporotic bone marrow culture conditions, involving response to differentiation medias, analysis of adipose and bone markers and cell migration in spheroid culture will be displayed. Quantitative and qualitative results, including fluorescence microscopy and in cell western, assessing the monolayer and spheroid cultures will be presented. The development of a pseudo osteoporosis model from healthy bone marrow will also be discussed. This model will form a basis of future work on miRNA targeting.

Osteoporosis is a global health issue with 200 million people suffering worldwide and it is a common condition in the elderly. Bisphosphonates including alendronate and risendronate are considered as the first line treatment for osteoporosis. However, there is increasing evidence that bisphosphonate (BP) therapy is associated with atypical fractures. Animal studies have reported a dose-dependent association between the duration of BP therapy and the accumulation of micro-damage. We tested the hypothesis that hip fracture patients treated with BP exhibited greater micro-damage density than untreated fracture and ‘healthy’ aging non-fracture controls.

Trabecular bone cores from patients treated with BP were compared with patients who had not received any treatment for bone metabolic disease (ethics reference: R13004). Non-fractured cadaveric femora from individuals with no history of bone metabolic disease were used as controls. Cores were imaged in high spatial resolution (∼1.3µm) using Synchrotron X-ray tomography (Diamond Light Source Ltd.) A novel classification system was devised to characterise features of micro-damage in the Synchrotron images: micro-cracks, diffuse damage and perforations. Synchrotron micro-CT stacks were visualised and analysed using ImageJ, Avizo and VGStudio MAX.

Our findings show that the BP group had the highest micro-damage density across all groups. The BP group (7.7/mm³) also exhibited greater micro-crack density than the fracture (4.3/mm³) and non-fracture (4.1/mm³) controls. Furthermore, the BP group (1.9/mm³) demonstrated increased diffuse damage when compared to the fracture (0.3/mm³) and non-fracture (0.8/mm³) controls. In contrast, the BP group (1.9mm³) had fewer perforations than fracture (3.0/mm³) and non-fracture controls (3.9/mm³).

BP inhibits bone remodelling, thereby reducing the number of perforated trabeculae, but over-suppression leads to micro-damage accumulation. Accumulated damage could weaken the trabecular bone in the femoral head and neck, increasing the risk of a fracture during a trip or fall.