Objectives. The role of proteinase activated receptor2 (PAR2) in regulating inflammation and tissue remodelling was evaluated in two experimental models of osteoarthritis (OA). Methods. OA was induced by either destabilisation of the medial meniscus (DMM) or disruption of the anterior cruciate ligament (ACL) in PAR2 wild-type (WT) and PAR2 knock-out (KO) mice (1). Using histological scoring and computerized planimetry, the effect of PAR2 deficiency on cartilage damage, bone sclerosis, and ligament remodelling was assessed at 1 year post-DMM induction. Synovitis and immune cell characterization were evaluated at 4 weeks post-surgery. Flow cytometry was used to assess immune cell populations in spleens from both models. Results. PAR2 KO mice showed a significant decrease in cartilage damage, bone sclerosis and ligament remodelling in one year DMM compared to WT (n=5 per group, p<0.05). Further characterization of the meniscotibial and anterior cruciate ligament in controls demonstrated the presence of PAR2, bone remodelling markers, hypertrophic-like chondrocyte cells and chondrocytelike cells, with an absence of macrophages. At 4 week in both WT DMM and ACL, macrophages (F4/80) were observed in the ligaments. Immune cell populations remained unchanged between groups with no significant synovitis observed. Conclusion. Disruption of PAR2 offers protection against cartilage degradation, bone sclerosis and ligament remodelling. There was no detectable inflammation in the OA models studied. 1. Glasson, SS et al. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage (2007), 15(9):1061-9. Titanium alloy with nanotubular surface modification for orthopaedic applications Eva Jablonská, Penelope M. Tsimbouri, Matthew J. Dalby Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic Centre for Cell Engineering, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Scotland eva.jablonska@vscht.cz Introduction Titanium and its alloys have been widely used in orthopaedics due to their good mechanical properties and biocompatibility. In order to accelerate healing and to improve the integration of the implant, various surface treatments can be performed. One of the possibilities is in situ preparation of nanotubes via anodic oxidation in the presence of fluoride ions. Nanostructured surfaces can mimic the extracellular matrix ability to trigger specific cell responses. Our aim is to prepare and test nanoscale surface modification that can support osteodifferentiation of the bone mesenchymal stem cells migrating to the site of the damage when implanted to the body. This would then prevent undesirable fibrous layer formation on the interphase leading to better fixation of the implant. Experimental Method The Ti-6Al-4V alloy with nanotubular surface modification (nanotubes of 50 nm and 100 nm in diameter) was tested using human mesenchymal stromal stem cells, human osteoprogenitors and human osteoblasts. Adhesion of MSCs and osteoprogenitors after three days was studied using fluorescent microscopy after immunostaining for vinculin and tubulin. Morphology of the cells and interaction with the nanotopography after three days was observed under scanning electron microscope. Metabolic activity of primary osteoblasts after one week was evaluated using MTT assay. After four weeks, markers of differentiation were determined using qPCR and morphology and cell numbers of MSCs were evaluated using immunofluorescent microscopy. Results We observed that adhesion and morphology on the surfaces was dependent on the cell type (MSCs being more sensitive than osteoprogenitors). Vinculin and tubulin intensity was significantly lower on nanostructured surfaces compared to polished control in case of the first donor; however, in the second case with different donor, there was no significant difference. Although there was neither significant difference in cell numbers nor in metabolic activity on the tested materials compared to polished control, we could observe a trend that nanotubes of smaller diameter (50 nm) seemed to be more suitable than 100nm nanotubes. Using qRT-PCR, we were not able to detect any conclusive changes in expression of osteogenic markers. ALP expression was downregulated on both nanosurfaces, whereas OCN expression was upregulated on 100nm nanotubes. Other osteogenic markers (OPN, OSX, osteonectin) as well as other markers of adipoand chondrodifferentiation (PPAR-gama and SOX9, respectively) remained unchanged. After four weeks of growth on the surfaces, MSCs on nanostructure had similar morphology to the polished control and there was no difference in cell numbers within the samples. Conclusions Nanotubular morphology on titanium alloy did not impair growth of primary human osteoblasts, osteoprogenitors and hMSCs. However we didn’t observe any strong effect of nanotubes on stem cell fate, which could be caused by interference of the fibrogenic character of the titanium material. Nanotubes of smaller diameter might have more pronounced effect. References 1. Dalby M. J., Gadegaard N., Oreffo R. O. C.: Nat. mater., 13, 558-569 (2014). This work was supported by EPSRC Analysis of osteoclastogenesis/osteoblastogenesis using human bone marrow derived co-cultures on nanotopographical titania surfaces. P. M.Tsimbouri, R.K. Silverwood, P. Fairhurst, T. Sjostrom, B. Su, P. Young, R.D.M. Meek, M.J. Dalby Centre for Cell Engineering, University of Glasgow, UK.Southern General Hospital, Glasgow, UK. School of Oral and Dental Science, University of Bristol, UK. INTRODUCTION: Titanium(Ti) an orthopaedic applications material with excellent load bearing properties. However, Ti is bioinert and this can affect osseointegration and outcomes of implants. Nanopatterning of implant surfaces could be the solution to this hurdle. We have shown that 15nm high nanopillars are bioactive using human MSCs and could improve osseointegration. We have also developed an osteoblast/osteoclast co-culture system using nanopits on polycarbonate. Here, we have used these co-cultures as they give an accurate representation of the in vivo environment, allowing assessment of bone remodelling related to biomaterials. AIM: Under co-culture conditions 15nm high Ti nanopillars, will induce osteoblastogenesis and reduce osteoclast activity, producing a method of enhancing secondary implant fixation. METHODS: Osteoblast/osteoclast progenitors co-cultured on polished Ti (control) and 15nm Ti nanopillars fabricated by the block copolymer technique. 14d and 28d time points were selected for analysis of osseointegration in vivo. Quantification of osteoclasts and bone nodule formation performed using histochemical staining. Morphological changes were examined by SEM. qRT-PCR was used to quantify expression of osteoblast, osteoclast and inflammatory response related genes. RESULTS: Using SEM, nanopillar Ti substrates were less inductive of osteoclastogenesis, with decreased maturity and activity with time, when compared to control (Fig.1a). Supportive TRAP staining showed macrophages/osteoclast projenitors present on control only (Fig.1b). Alizarin red staining indicated increased osteogenesis on the nanopillars (Fig.1c). qRT-PCR revealed a timerelated decrease in osteoclastogenesis-related genes on the nanopillars with an associated increase in osteoclast inhibitors. Fig.1 Showing a) SEM, b) TRAP and c) Alizarin staining results (n=3, T-test, *p<0.05, ***p<0.001). DISCUSSION: Dramatic reduction in number of osteoclast progenitor cells and increased osteoblastogenesis on the nanopillar substrates versus control. Genomic data on osteoclast and osteoblast-related genes will be presented. REFERENCES: L.E. McNamara,et al (2012) J. R. Soc. Interface. T. Sjöström et al (2013) Adv. Healthcare Mater. P. Young et al., (2014), Nanomedicine. ACKNOWLEDGMENTS: EPSRC funded. HLA-B27 over-expression in rats alters central and peripheral monocyte populations. Cecilia Ansalone, Simon Milling, Carl S. Goodyear Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary Medicine and Life Sciences, University of Glasgow, Glasgow, UK Background and objectives: HLA-B27 expression is associated with spondyloarthropathy (SpA) and transgenic rats expressing human HLA-B27 and b2-microglobulin (B27 rats) display systemic inflammation and bone loss, which resembles human SpA. We have previously demonstrated that these rats lack a dendritic cell population. However, the myeloid compartment has not been completely characterized. Here, we aimed to characterize monocyte and pre-osteoclast populations in the bone marrow and blood of B27 rats, and examined the potential of different monocyte/preosteoclast subsets to generate mature osteoclasts in vitro. Materials and methods: 14-16 week old control (B7) and B27 rats were bled and plasma CCL2 levels were measured by ELISA. Monocytes subsets were analysed and quantified in both the bone marrow and blood of B27, B7 and non-transgenic animals, by evaluation of surface markers (CD172a, CD43, and CD11b) and intracellular uptake of fluorescent M-CSF and CCL2 by flow cytometry. Monocyte populations were FACS sorted and cultured in pro-osteoclastogenic medium for 7 days to evaluate osteoclastogenic potential. Cultures were stained with tartrate-resistant acid phosphatase (TRAP) and mature osteoclasts (TRAP and ≥3 nuclei) were quantified by light microscopy. Results: A previously unidentified CD172a CD43 CD11b population of monocytes was observed in the bone marrow. These are bona fide monocytes, expressing CD115 and CCR2, as determined by uptake of fluorescent M-CSF and CCL2 respectively. Interestingly, this new monocyte population was significantly increased in B27 rats. Assessment of the osteoclastogenic potential of bone marrow monocyte subsets revealed that CD172a CD43 CD11b, but not CD172a CD43 CD