Human mesenchymal stem cells are considered the golden standard for clinical application in regenerative medicine for their multilineage differentiation potential, best candidates to treat diseases such as osteoarthritis and osteogenesis imperfecta. In the past few years several molecules have been described to induce the hMSCs differentiation into osteo cell progenitors, mainly discovered by screening of single metabolites bioactivity. However, hMSCs osteogenic differentiation potential is still poor, and the discovery of differentiation inducers with high efficiency is needed. Thanks to automated processes, High Throughput Screening (HTS) strategies shorten the metabolites bioactivity investigation timeline, allowing testing of many molecules simultaneously. In this work, reliable assays for natural products bioactivity investigation detection were developed using HTS methodologies and validated by testing 15 purified compounds derived by marine fungi and sponges. The HTS cytotoxicity investigation using HepG2 cells allowed to test in a single experiment, 15 metabolites in 4 concentrations ranging from 1 to 20µM. Low cytotoxicity was detected for metabolites concentrations from 1 to 10µM and so set as treatment concentrations to be tested in further assays. Anti-inflammatory bioactivity was tested on THP1 cells triggered by LPS. Five out of 15 metabolites showed to prevent the LPS induced THP1 inflammatory activation by lowering the TNF-α production. The metabolites pro-osteogenic potential was investigated using hMSCs: their differentiation was evaluated by calcium mineralization after 10 days differentiation. Pro-osteogenic molecules were not detected in this screening, but the method validation represents a powerful tool for future natural product and synthetic molecules libraries screenings

Currently implemented accuracy metrics in open-source libraries for segmentation by supervised machine learning are typically one-dimensional scores [1]. While extremely relevant to evaluate applicability in clinics, anatomical location of segmentation errors is often neglected. This study aims to include the three-dimensional (3D) spatial information in the development of a novel framework for segmentation accuracy evaluation and comparison between different methods. Predicted and ground truth (manually segmented) segmentation masks are meshed into 3D surfaces. A template mesh of the same anatomical structure is then registered to all ground truth 3D surfaces. This ensures all surface points on the ground truth meshes to be in the same anatomically homologous order. Next, point-wise surface deviations between the registered ground truth mesh and the meshed segmentation prediction are calculated and allow for color plotting of point-wise descriptive statistics. Statistical parametric mapping includes point-wise false discovery rate (FDR) adjusted p-values (also referred to as q-values). The framework reads volumetric image data containing the segmentation masks of both ground truth and segmentation prediction. 3D color plots containing descriptive statistics (mean absolute value, maximal value,…) on point-wise segmentation errors are rendered. As an example, we compared segmentation results of nnUNet [2], UNet++ [3] and UNETR [4] by visualizing the mean absolute error (surface deviation from ground truth) as a color plot on the 3D model of bone and cartilage of the mean distal femur. A novel framework to evaluate segmentation accuracy is presented. Output includes anatomical information on the segmentation errors, as well as point-wise comparative statistics on different segmentation algorithms. Clearly, this allows for a better informed decision-making process when selecting the best algorithm for a specific clinical application

Precision health aims to develop personalised and proactive strategies for predicting, preventing, and treating complex diseases such as osteoarthritis (OA). Due to OA heterogeneity, which makes developing effective treatments challenging, identifying patients at risk for accelerated disease progression is essential for efficient clinical trial design and new treatment target discovery and development. To create a reliable and interpretable precision health tool that predicts rapid knee OA progression over a 2-year period from baseline patient characteristics using an advanced automated machine learning (autoML) framework, “Autoprognosis 2.0”. All available 2-year follow-up periods of 600 patients from the FNIH OA Biomarker Consortium were analysed using “Autoprognosis 2.0” in two separate approaches, with distinct definitions of clinical outcomes: multi-class predictions (categorising disease progression into pain and/or radiographic progression) and binary predictions. Models were developed using a training set of 1352 instances and all available variables (including clinical, X-ray, MRI, and biochemical features), and validated through both stratified 10-fold cross-validation and hold-out validation on a testing set of 339 instances. Model performance was assessed using multiple evaluation metrics. Interpretability analyses were carried out to identify important predictors of progression. Our final models yielded higher accuracy scores for multi-class predictions (AUC-ROC: 0.858, 95% CI: 0.856-0.860) compared to binary predictions (AUC-ROC: 0.717, 95% CI: 0.712-0.722). Important predictors of rapid disease progression included WOMAC scores and MRI features. Additionally, accurate ML models were developed for predicting OA progression in a subgroup of patients aged 65 or younger. This study presents a reliable and interpretable precision health tool for predicting rapid knee OA progression. Our models provide accurate predictions and, importantly, allow specific predictors of rapid disease progression to be identified. Furthermore, the transparency and explainability of our methods may facilitate their acceptance by clinicians and patients, enabling effective translation to clinical practice

Abstract. Objectives. A promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect. Methods. Four weeks after a medial meniscus transection surgery, sheep were injected with 10. 7. hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMiner. TM. ) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses. Results. hUC-MSC treated animals demonstrated significantly less joint space narrowing. Nineteen SF proteins were differentially abundant in treated cf. control sheep (FC±2.0; p<0.05). Biglycan (a small leucine-rich proteoglycan of the cartilage extracellular matrix) abundance was increased by 2.1 fold in treated compared to untreated sheep (p=0.024). IPA indicated that lipid synthesis (z-score=1.772; p=0.00267) and immune cell migration pathways (cell movement of mononuclear leukocytes: z-score=1.761; p=0.00259), amongst others, were likely to be activated in the treated sheep. Conversely, tissue damage (z-score=−2; p=0.00019), senescence (z-score=−1.981; p=0.00007) and necrosis (z-score=−1.728; p=0.00829) associated pathways as well as inflammation (z-score=−1.718; p=0.00057) and vascular permeability (z-score=−1.698; p=0.00002) were likely to be inhibited in treated cf. untreated sheep. Conclusions. hUC-MSC treatment prevented/delayed OA progression, demonstrated via a reduction in joint space narrowing. SF proteome bioinformatics revealed potential mechanisms of therapeutic action related to immunomodulation and the inhibition of multiple cell death, and tissue damage associated pathways. Further, a potential predicted upregulation in lipid synthesis in treated sheep represents a novel mechanism warranting further investigation. Additional work is required to validate these discovery phase proteomic findings in studies which specifically target and manipulate the proposed mechanisms highlighted. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Introduction. Precision health aims to develop personalised and proactive strategies for predicting, preventing, and treating complex diseases such as osteoarthritis (OA), a degenerative joint disease affecting over 300 million people worldwide. Due to OA heterogeneity, which makes developing effective treatments challenging, identifying patients at risk for accelerated disease progression is essential for efficient clinical trial design and new treatment target discovery and development. Objectives. This study aims to create a trustworthy and interpretable precision health tool that predicts rapid knee OA progression based on baseline patient characteristics using an advanced automated machine learning (autoML) framework, “Autoprognosis 2.0”. Methods. All available 2-year follow-up periods of 600 patients from the FNIH OA Biomarker Consortium were analysed using “Autoprognosis 2.0” in two separate approaches, with distinct definitions of clinical outcomes: multi-class predictions (categorising patients into non-progressors, pain-only progressors, radiographic-only progressors, and both pain and radiographic progressors) and binary predictions (categorising patients into non-progressors and progressors). Models were developed using a training set of 1352 instances and all available variables (including clinical, X-ray, MRI, and biochemical features), and validated through both stratified 10-fold cross-validation and hold-out validation on a testing set of 339 instances. Model performance was assessed using multiple evaluation metrics, such as AUC-ROC, AUC-PRC, F1-score, precision, and recall. Additionally, interpretability analyses were carried out to identify important predictors of rapid disease progression. Results. Our final models yielded high accuracy scores for both multi-class predictions (AUC-ROC: 0.858, 95% CI: 0.856–0.860; AUC-PRC: 0.675, 95% CI: 0.671–0.679; F1-score: 0.560, 95% CI: 0.554–0.566) and binary predictions (AUC-ROC: 0.717, 95% CI: 0.712–0.722; AUC-PRC: 0.620, 95% CI: 0.616–0.624; F1-score: 0.676, 95% CI: 0.673–0679). Important predictors of rapid disease progression included the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores and MRI features. Our models were further successfully validated using a hold-out dataset, which was previously omitted from model development and training (AUC-ROC: 0.877 for multi-class predictions; AUC-ROC: 0.746 for binary predictions). Additionally, accurate ML models were developed for predicting OA progression in a subgroup of patients aged 65 or younger (AUC-ROC: 0.862, 95% CI: 0.861–0.863 for multi-class predictions; AUC-ROC: 0.736, 95% CI: 0.734–0.738 for binary predictions). Conclusions. This study presents a reliable and interpretable precision health tool for predicting rapid knee OA progression using “Autoprognosis 2.0”. Our models provide accurate predictions and offer insights into important predictors of rapid disease progression. Furthermore, the transparency and interpretability of our methods may facilitate their acceptance by clinicians and patients, enabling effective utilisation in clinical practice. Future work should focus on refining these models by increasing the sample size, integrating additional features, and using independent datasets for external validation. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Objectives. Direct ink writing (DIW) has gained considerable attention in production of personalized medical implants. Laponite nanoclay is added in polycaprolactone (PCL) to improve printability and bioactivity for bone implants. The 3D structure of DIW printed PCL/Laponite products was qualitatively evaluated using micro-CT. Methods. PCL/LP composite ink was formulated by dissolving 50% m/v PCL in dichloromethane with Laponite loading of up to 30%. The rheological properties of the inks were determined using Discovery HR-2 rheometer. A custom-made direct ink writer was used to fabricate both porous scaffold with 0°/90° lay-down pattern, and solid dumbbell-shaped specimens (ASTM D638 Type IV) with two printing orientations, 0° and 90° to the loading direction in tensile testing. The 3D structure of specimens was assessed using a micro-CT. Independent t-tests were performed with significance level at p<0.05. Results. The addition of Laponite in PCL ink has significantly enhanced viscosity for shape fidelity and shear-thinning property facilitating extrusion for DIW. Uniform distribution of Laponite was illustrated by micro-CT. For the 32-layer scaffold, interconnectivity of pores is observed at all 3 planes. The variation of height and width of layers is within 6% except the bottom 2 layers which are significantly lower and wider than other layers for mechanical support. For solid specimens, no ditches/interfaces between filaments are observed in 90° orientation while they are distinctive in 0° orientation because deposited filaments contact each other sooner in 90° orientation. 90° specimens also have lower air gap fraction (0.8 vs 5.4 %) and significantly higher Young's modulus (235 vs 195 MPa) and tensile strength (12.0 vs 9.5 MPa). Conclusions. The mechanical properties and printability of PCL/Laponite composites can be improved by controlling printing parameters; Micro-CT is an important tool to investigate the structure and properties of 3D printed products for bone tissue engineering

Introduction and Objective. Home-based monitoring of fracture healing has the potential of reducing routine follow-up and improve personalized fracture care. Implantable sensors measuring electrical impedance might detect changes in the electrical current as the fracture heals. The aim was to investigate whether electrical impedance correlated with radiographic fracture healing. Materials and Methods. Eighteen rabbits were subjected to a tibial osteotomy that was stabilized with an external fixator. Two electrodes were positioned, one electrode placed within the medullary cavity and the other on the lateral cortex, both three millimeters from the osteotomy site. Transverse electrical impedance was measured daily across the fracture site at a frequency range of 5 Hz to 1 MHz using an Analog Discovery 2 Oscilloscope with Impedance Analyzer. Biweekly x-rays were taken and analyzed blinded using a modified anterior-posterior (AP) radiographic union score of the tibia (RUST). Each animal served as its own control by performing repeated measurements from time zero until the end of follow-up. Results. At 5 Hz measurements, a linear mixed model revealed an average impedance at day zero of 10670 +/− 272 Ohm (p<0.001) and a change in impedance from day 0 to day 7 of −3330 +/− 152 (p<0.001). The slope from day 0–7 was estimated as −548.6 +/− 26 (p<0.001) and was steeper than the slope after day 7 which was estimated to −85.6 +/− 4 (p<0.001). This indicates that the impedance decreased quicker before day 7 and slower after day 7. The coefficient of variation for difference between RUST scores, from double intra-rater measurements of 15 radiographs with a minimum of 22 days between, was 1.3. Spearman's correlation coefficient between impedance and RUST score at the 5 Hz was −0.75 (p<0.001). Conclusions. This osteotomy model showed that the electrical impedance can be measured in vivo at a distance from the fracture site with a consistent change in impedance over time. This is the first study to demonstrate a significant correlation between increasing radiographic union score and decreasing impedance. Further studies are warranted to investigate how these new and important results can further be translated into larger animal studies

Summary Statement. Discovery system produced effective functional improvement in both primary and revision total elbow replacement. The incidence of major complications was in an acceptable range. Introduction. The search for the ideal elbow prosthesis continues as instability and loosening remain the prime reasons for total elbow replacement (TER) failure. The Discovery Elbow System (Biomet) is one of the latest generations of linked prosthesis and has been used in UK since 2003. We report outcome of TER using this system. Methods. A total of 100 TERs (75 primary, 25 revisions) were performed between 2003 and 2010. The main primary underlying pathologies for TER were advanced rheumatoid arthritis (N=58), osteoarthritis (N=35), acute fractures (N=7). There were 60 female and 40 male patients with an average age of 62 years. The outcome assessment included pain, patient satisfaction, Liverpool Elbow Score (LES), range of movement, and imaging during a mean follow-up period of 48.5 months. Major complications are also reported. Results. For the whole patient group (primary + revision), the LES was significantly (p<0.001) improved from 3.79+/−1.71 to 6.36+/−1.85There were significant improvements in elbow flexion from 100°+/−24 to 118°+17, supination from 38°+/−26 to 50°+/−25 and pronation from 48°+/−22 to 61°+/−21. Mean improvement in flexion-extension and pronation-supination arc was 20° and 25°, respectively. 64% of cases were completely pain-free and at the final follow-up (compared to 7% preoperatively). Only 6% of patients scored “Not Satisfied” at the final follow-up. LES improvement was significantly higher in the primary TER compared to revision TER (p<0.05). Imaging reviewed for 60 cases showed loosening in 4% of patients. Other main complications included deep infection (N=2), ulnar neuropathy (N=3), pre-prosthetic fracture (N=2), and prosthetic failure (N=1). Discussion. TER using the Discovery Elbow System is an effective arthroplasty in terms of functional improvement, pain relief and range of motion in both primary and revision patients. TER resulted in no/mild pain in 78% of cases. Patients undergoing Acclaim, Souter-Strathclyde, GSB III, and Coonrad-Morrey TER have been reported to have no/mild pain in 64%, 67%, 50–92% and 60–100% of cases, respectively. A 20° improvement in flexion-extension arc is comparable to that of Acclaim (23°), Souter-Strathclyde (15°), GSBIII (19–33°), and Coonrad-Morrey (17–26°) TER. An improvement of 25° in pronation-supination arc in our series is also comparable to that of 21–28° reported the Coonrad-Morrey and 27–33° for Discovery prostheses. An infection rate of 2% is lower than several other reports for GSB III TER (7–11%) and Coonrad-Morrey (6–8%). The incidence of persistent ulnar neuropathy (3%) was lower compared to GSBIII TER (11–14%), Coonrad-Morrey (12–26%), and Acclaim (8%)

Heterotopic ossification (HO) is lamellar bone formation that occurs within tissues that do not normally have properties of ossification. The pathoaetiology of HO is poorly understood. We conducted a genome wide association study to better understand the genetic architecture of HO. 891 patients of European descent (410 HO cases) following THA for primary osteoarthritis were recruited from the UK. HO was assessed from plain AP radiographs of the pelvis. Genomic DNA was extracted, genotyped using the Illumina 610 beadchip and referenced using the 1000 Genome Project panel. HO susceptibility case-control analysis and an evaluation of disease severity in those with HO was undertaken using SNPTESTv2.3.0 on>10 million variants. We tested variants most strongly associated with HO in an independent UK THA replication cohort comprising 209 cases and 211 controls. The datasets were meta-analysed using PLINK. In the discovery cohort 70 signals with an index variant at p<9×10–5 were suggestively associated with HO susceptibility. The strongest signal lay just downstream of the gene ARHGAP18 (rs59084763, effect allele frequency (EAF) 0.19, OR1.87 [1.48–2.38], p=2.48×10–8), the second strongest signal lay within the long non-coding (LNC) RNA gene CASC20 (rs11699612, EAF 0.25, OR1.73 [1.1.40–2.16, p=9.3×10–8). In the discovery cohort 73 signals with an index variant at p<9×10–5 were associated with HO severity. At replication, 12 of the leading 14 susceptibility signals showed a concordant direction of allelic effect and 5 replicated at nominal significance. Following meta-analysis, the lead replicating susceptibility signal was the CASC20 variant rs11699612 (p=2.71×10–11). We identify consistent replicating association of variation within the LNC RNA CASC20 with HO susceptibility after THA. Although the function of CASC20 is currently unknown, possible mechanisms include transcriptional, post-transcriptional and epigenetic regulation of downstream target genes. The work presented here provides new avenues for the development of novel predictive and therapeutic approaches towards HO

More than 250,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US, with a cost of $17–25K/patient. There is an unmet clinical demand for improving grafts/scaffolds to provide biological integration in addition to mechanical support. Currently, no data is available for the utilization of fibrous scaffolds with bimodal distribution for ACL regeneration. The novelty in this study is that it proposes for the first time to investigate the collagen fibril diameter distribution in healthy and injured bovine ACL tissue, and utilization of such structure for scaffold design. Objectives are 1) developing a bovine ACL tear model and measuring the collagen fibril diameter distribution of both healthy and injured ACL tissues, and 2) fabricating scaffolds to mimic the structural properties of healthy and injured ACL tissue. Bovine ACL tissues (1–3 years old) were harvested and characterized for their fibril diameter distribution using Transmission Electron Microscopy (TEM) and biomechanical properties under tension. The electrospun polycaprolactone (PCL) scaffolds were characterized using SEM and mechanical testing. Healthy and injured ACL fibril diameter, and that of PCL scaffolds representing healthy and injured ACL are compared using unpaired student t-test. The proposed fibrous scaffold design represents a significant departure from the conventional unimodal approach, and is expected to have significant contribution to ACL regeneration. These discoveries will serve as the foundation for the development of biomimetic tissue engineering substrates aimed at promoting biological graft fixation

Objectives. Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods. A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results. This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion. This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1

Abstract. OBJECTIVES. Staphylococcus aureus is one of the most common pathogens in orthopaedic biomaterial-associated infections. The transition of planktonic S. aureus to its biofilm phenotype is critical in the pathogenesis of biomaterial-associated infections and the development of antimicrobial tolerance, which leads to ineffective eradication in clinical practice. This study sought to elucidate the effect of non-lethal dispersion on antimicrobial tolerance in S. aureus biofilms. METHODS. Using a methicillin-sensitive S. aureus reference strain, the effect of non-lethal dispersion on gentamicin tolerance, cellular activity, and the intracellular metabolome of biofilm-associated bacteria were examined. Gentamicin tolerance was estimated using the dissolvable bead biofilm assay. Cellular activity was estimated using the triphenyltetrazolium chloride assay. Metabolome analysis was performed using tandem high-performance liquid chromatography and mass spectrometry. RESULTS. Non-lethal dispersion of biofilm-associated S. aureus was associated with a four-fold reduction in gentamicin tolerance and a 25% increase in cellular respiration of both dispersed and adherent cells. Metabolome analysis found non-lethal dispersion reduced intracellular levels of L-ornithine and L-proline, with increased levels of cyclic nucleotides (p<0.05) in both liberated cells and the remaining biofilm-associated bacteria. These metabolomic changes have previously been shown to be associated with inactivation of the carbon catabolite repression mechanism, which is a key regulatory gatekeeper in the cellular resuscitation of dormant S. aureus cells. CONCLUSION. The metabolomic pipeline described in this study presents a valuable tool in the elucidation of molecular mechanistic pathways in biofilm pathogenesis. Kreb's cycle reactivation, through the carbon catabolite repression regulatory mechanism, has been shown to be associated with the reversal of biofilm-associated gentamicin tolerance. Understanding of the biosynthetic changes associated with the biofilm state will assist in the discovery of novel therapeutic targets in the management of biomaterial-related infections. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Summary Statement. Developing titanium (Ti) surfaces that are biocompatible yet serve as deterrents for bacterial attachment and growth are particularly appealing in tackling the ongoing problem of sepsis-induced implant failures. Realising this could include coating Ti with the bioactive lipid, lysophosphatidic acid. Introduction. Surgical revision for failed total joint replacements costs a staggering £300m/yr and approximately 20% of this burden is attributed to implant failure through bacterial infection. Producing biomaterials that deter microbial attachment as well as securing robust osseointegration continues to be a significant research challenge in contemporary bone biomaterials design. Steps to realising novel improvements are further compounded by the concerns raised over resistance of bacteria to many antimicrobial agents. Clearly this is a major constraint necessitating an entirely novel approach to minimising implant infection risk. We therefore turned our attention to certain lysophosphatidic acids (LPAs) for Ti functionalisation. We have found LPA to enhance calcitriol-induced human osteoblast (hOB) maturation. Of further significance is the discovery that LPA can directly inhibit the growth of certain bacteria and even co-operate with some antibiotics to bring about their demise. Herein we describe the fabrication of a hOB-compatible Ti surface with palmitoyl-LPA (P-LPA) which we also find hinders bacterial attachment. Methods. We adopted a self-assembly strategy for the attachment of P-LPA to Ti. Briefly Ti discs (Corin Group, Cirencester, UK) were baked, overnight, at 160°C and then coated with octadecylphosphonic acid (ODP) which has a natural affinity for Ti oxide. Bound ODP provided a tethering point for P-LPA via hydrophobic interaction with the “tail” region perpendicular to the Ti surface. Modified Ti discs were subsequently seeded with hOBs to evaluate their maturation response to calcitriol. In addition modified Ti samples were exposed to either Staphylococcus epidermidis or methicillin-resistant Staphylococcus aureus and the extent of surface coverage determined via crystal violet staining following 24hr incubation. Results. The development of P-LPA functionalised Ti provided a surface that secured hOB maturation in response to calcitriol, as supported by significant increases in total alkaline phosphatase activity, an enzyme expressed in greater abundance as hOBs progress to a more differentiated phenotype. In contrast this Ti substrate was not as attractive to bacteria as evaluated by crystal violet staining and dye recovery from the incubated specimens. Discussion. Multifunctional bone biomaterials that combine host tissue biocompatibility with an antibacterial surface finish will represent the next-generation orthopaedic devices. The biologically active lysophospholipid, LPA, is assuming an emerging interest in hOB biology. This has partly arisen from our discovery that it co-operates with calcitriol to bolster the formation and maturation of hOBs. Another equally exciting property of LPA is the discovery that it can inhibit the growth of bacteria and, in some instances, co-operate with certain antibiotics in killing bacteria. The application of ODP for the attachment of P-LPA to Ti presented itself as a facile step towards developing a novel Ti surface finish. Collectively our preliminary investigations indicate that our modified Ti supports calcitriol-induced hOB maturation but that it deters bacteria

Early detection of knee osteoarthritis (OA) is critical for possible preventive treatment, such as weight loss, physical activity and sports advice and restoring biomechanics, to postpone total knee arthroplasty (TKA). Specific biomarkers for prognosis and early diagnosis of OA are lacking. Therefore, in this study, we analyzed the lipid profiles of different tissue types within Hoffa's fat pad (HFP) of OA and cartilage defect (CD) patients, using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). The HFP has already been shown to play an important role in the inflammatory process in OA by prostaglandin release. Additionally, MALDI-MSI allows us to investigate on tissue lipid distribution at molecular level, which makes it a promising tool for the detection of disease specific biomarkers for OA development. Samples of HFP were obtained of patients undergoing surgical treatment for OA (n=3) (TKA) or CD (n=3) (cartilage repair). In all cases, tissue was obtained without patient harm. HFP samples were washed in phosphate buffered saline (PBS) and snap-frozen directly after surgical dissection to remove redundant blood contamination and to prevent as much tissue degradation as possible. Tissue sections were cut at 15 µm thickness in a cryostat (Leica Microsystems, Wetzlar) and deposited on indium tin oxide glass slides. Norharmane (Sigma-Aldrich) matrix was sublimed onto the tissue using the HTX Sublimator (HTX Technologies, Chapel Hill). µMALDI-MSI was performed using Synapt G2Si (Waters) at 50 µm resolution in positive ion mode. MS/MS fragmentation was performed for lipid identification. Data were processed with in-house Tricks for MATLAB and analyzed using principle component analysis (PCA) and verlan. OA and CD HFP specific lipid profiles were revealed by MALDI-MSI followed by PCA and DA. With these analyses we were able to distinguish different tissue types within HFP of different patient groups. Further discriminant analysis showed HFP intra-tissue heterogeneity with characteristic lipid profiles specific for connective and adipose tissues, but also for synovial tissue and blood vessels, revealing the high molecular complexity of this tissue. As expected, lipid signals were lower at the site of the connective tissue, compared to the adipose tissue. In particular, tri-acyl glycerol, di-acyl glycerol, sphingomyelin and phosphocholine species were differently abundant in the adipose tissue of HFP of OA compared to CD. To our knowledge, this is the first study comparing lipid profiles in HFP of OA patients with CD patients using MALDI-MSI. Our results show different lipid profiles between OA and CD patients, as well as intra-tissue heterogeneity within HFP, rendering MALDI-MSI as a useful technology for OA biomarker discovery. Future research will focus on expanding the number of subjects and the improvement of lipid detection signals

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502

Objectives. In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation. Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594–601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1

Currently, there are no generally accepted treatments for the prevention of osteonecrosis. To compound this further, despite considerable research efforts, the natural history of this disease remains poorly understood. The disease process appears to be initially asymptomatic, but after symptoms appear, the course becomes rapidly progressive. Clinical studies have shown that, if left untreated, collapse of the femoral head will occur in 80 per cent of the cases or greater within four years. As our knowledge of the etiology and pathogenesis of osteonecrosis improves, new treatments to halt, or at least impede, the progression of the disease may be possible. Achieving the best outcomes in the treatment of osteonecrosis depends on early, accurate diagnosis, and prompt treatment appropriate for the stage of the disease. In many cases, if treated early, long-term preservation of the native joint is possible. Magnetic resonance imaging allows accurate diagnosis in even the earliest asymptomatic stages of the disease. Non-surgical treatments such as pharmacological agents have shown promise in experimental studies, although further work remains before they are appropriate for widespread use. Various hip salvaging procedures such as core decompression, percutaneous drilling, non-vascularized and vascularized bone grafting, and various osteotomies have been successful in the majority of properly selected patients over follow-up times of a decade or more. Advances in arthroplasty technologies and techniques, including hip resurfacing and modern cementless total hip arthroplasty have allowed patients to return to pain-free, active lifestyles with excellent long-term prosthesis survival. Current treatments for osteonecrosis, while generally successful, focus on halting or delaying the progression of symptomatic disease. Recent discoveries concerning the relationship between genetic factors and the development of osteonecrosis, as well as the pathophysiologic effects of various indirect and direct risk factors such as corticosteroid use and sickle cell disease, continue to improve our understanding of the underlying disease process. While these discoveries are promising, we must continue to work towards the goal of being able to identify and treat the precursors of osteonecrosis before it progresses to symptomatic disease and threatens the survival of native joints

Summary Statement. A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction. Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo. Methods. hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global gene expression profiles were evaluated prior and after bioreactor culture. Bone development was investigated using biochemical and histological methods, and by micro-computed tomography (μCT) imaging over the duration of bioreactor culture and after 12-week subcutaneous implantation in immunodeficient mice. Results. Progenitors with high proliferation potential, expressing typical mesenchymal surface antigens were successfully derived from three hiPSC lines. Differences in mesenchymal surface antigens expression and global gene expression profiles of progenitors from different lines corresponded to their differentiation abilities toward the osteogenic, chondrogenic and adipogenic lineages. Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into the culture medium as compared to static culture. Dense bone matrix formation was evidenced by the positive staining of collagen, osteopontin, bone sialoprotein and osteocalcin. In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce. μCT revealed a significant increase in bone structural parameters, evidencing mineralization of the deposited bone tissue during the 5-week culture in bioreactors. Osteogenesis and bone tissue formation were comparable between hESCs, hiPSCs and hMSCs (3). Bioreactor cultivation resulted in repression of genes involved in proliferation and tumorigenesis, and upregulation of genes associated with osteogenesis and bone development. Engineered bone tissue displayed stable phenotype after 12-week implantation in vivo, with cells of human origin, ingrowing vasculature and osteoclasts, suggesting an initiation of tissue remodeling. Discussion/Conclusion. Our biomimetic strategy opens the possibility to construct an unlimited quantity of patient-specific bone grafts for personalised applications, and to generate qualified experimental models to study bone biology under normal and pathological conditions, as well as test new drugs using selected pools of hiPSC lines