Introduction. Intervertebral disc degeneration has been associated with low back pain (LBP) which is a major cause of long-term disability worldwide. Observed mechanical and biological modifications have been related to decreased water content. Clinical traction protocols as part of LBP management have shown positive outcomes. However, the underlying mechanical and biological processes are still unknown. The study purpose was to evaluate the impact of unloading through traction on the mechanobiology of healthy bovine tail discs in culture. Method. We loaded bovine tail discs (n=3/group) 2h/day at 0.2Hz for 3 days, either in dynamic compression (-0.01MPa to -0.2MPa) or in dynamic traction (-0.01MPa to 0.024MPa). In between the dynamic loading sessions, we subjected the discs to static compression loading (-0.048MPa). We assessed biomechanical and biological parameters. Result. Over the 3 days of loading, disc height decreased upon dynamic compression loading but increased upon unloading. The neutral zone was restored for all samples at the end of the dynamic unloading. Upon dynamic compression, the stiffness increased over time while the hysteresis decreased. Upon dynamic unloading, sulfated glycosaminoglycan (sGAG) release in the medium was lower at the endpoint. In the outer annulus fibrosus (AFo), we saw a higher water/sGAG of at least 30%. In the nucleus pulposus, COL2 mRNA was expressed more highly upon dynamic unloading while MMP3, iNOS and TRPV4 expression levels were lower. In the AFo of the unloading group, COL2 expression was higher but COL1 was lower. Conclusion. The biomechanical and biological results consistently indicate that dynamic unloading of healthy bovine discs in culture facilitates water uptake and promotes an anti-catabolic response which reflects a function optimization of the disc. This work combines biomechanical and biological results and opens the door to evidence-based improvement of regenerative protocols for degenerated discs and conservative LBP management. This study is funded by AO Foundation and AO Spine

Mincing cartilage with commercially available shavers is increasingly used for treating focal cartilage defects. This study aimed to compare the impact of mincing bovine articular cartilage using different shaver blades on chondrocyte viability. Bovine articular cartilage was harvested using a scalpel or three different shaver blades (2.5 mm, 3.5 mm, or 4.2 mm) from a commercially available shaver. The cartilage obtained with a scalpel was minced into fragments smaller than 1 mm. 3. All four conditions were cultivated in a culture medium for seven days. After Day 1 and Day 7, metabolic activity, RNA isolation, and gene expression of anabolic (COL2A1, ACAN) and catabolic genes (MMP1, MMP13), Live/Dead staining and visualization using confocal microscopy, and flow cytometric characterization of minced cartilage chondrocytes were measured. The study found that mincing cartilage with shavers significantly reduced metabolic activity after one and seven days compared to scalpel mincing (p<0.001). Gene expression of anabolic genes was reduced, while catabolic genes were increased after day 7 in all shaver conditions. The MMP13/COL2A1 ratio was also increased in all shaver conditions. Confocal microscopy revealed a thin line of dead cells at the lesion site with viable cells below for the scalpel mincing and a higher number of dead cells diffusely distributed in the shaver conditions. After seven days, there was a significant decrease in viable cells in the shaver conditions compared to scalpel mincing (p<0.05). Flow cytometric characterization revealed fewer intact cells and proportionally more dead cells in all shaver conditions compared to the scalpel mincing. Mincing bovine articular cartilage with commercially available shavers reduces the viability of chondrocytes compared to scalpel mincing. This indicates that mincing cartilage with a shaver should be considered a matrix rather than a cell therapy. Further experimental and clinical studies are required to standardize the mincing process with a shaver. Acknowledgements: This study received unrestricted funding from KARL STORZ SE & Co. KG

Miniscrew implants (MSIs) are widely used to provide absolute anchorage for the orthodontic treatment. However, the application of MSIs is limited by the relatively high failure rate (22.86%). In this study, we wished to investigate the effects of amorphous and crystalline biomimetic calcium phosphate coating on the surfaces of MSIs with or without the incorporated BSA for the osteointegration process with an aim to facilitate the early loading of MSIs. Amorphous and crystalline coatings were prepared on titanium mini-pin implants. Characterizations of coatings were examined by Scanning electron microscopy (SEM), Confocal laser-scanning dual-channel-fluorescence microscopy (CLSM) and Fourier-transform infrared spectroscopy (FTIR). The loading and release kinetics of bovine serum albumin (BSA) were evaluated by Enzyme linked immunosorbent assay (ELISA). Activity of alkaline phosphate (ALP) was measured by using the primary osteoblasts. In vivo, a model of metaphyseal tibial implantation in rats was used (n=6 rats per group). We had 6 different groups: no coating no BSA, no coating but with surface adsorption of BSA and incorporation of BSA in the biomimetic coating in the amorphous and crystalline coatings. Time points were 3 days, 1, 2 and 4 weeks. Histological and histomorphometric analysis were performed and the bone to implant contact (BIC) of each group was compared. In vitro, the incorporation of BSA changed the crystalline coating from sharp plates into curly plates, and the crystalline coating showed slow-release profile. The incorporation of BSA in crystalline coating significantly decreased the activity of ALP in vitro. In vivo study, the earliest significant increase of BIC appeared in crystalline coating group at one week. The crystalline coating can serve as a carrier and slow release system for the bioactive agent and accelerate osteoconductivity at early stage in vivo. The presence of BSA is not favorable for the early establishment of osteointegration

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

Introduction and Objective. Low back pain (LBP) is a major cause of long-term disability in adults worldwide and it is frequently attributed to intervertebral disc (IVD) degeneration. So far, no consensus has been reached regarding appropriate treatment and LBP management outcomes remain disappointing. Spine unloading or traction protocols are common non-surgical approaches to treat LBP. These treatments are widely used and result in pain relief, decreased disability or reduced need for surgery. However, the underlying mechanisms -namely, the IVD unloading mechanobiology- have not yet been studied. The aim of this first study was to assess the feasibility of IVD unloading in a large animal organ culture set-up and evaluate its impact on mechanobiology. Materials and Methods. Bovine tail discs (diameter 16.1 mm ± 1.2 mm), including the endplates, were isolated and prepared for culture. Beside the day0 sample that was processed directly, three other discs were cultured for 3 days and processed on day4. One disc was loaded in the bioreactor according to a previously established physiological (compressive) loading protocol (2h/day, 0.2Hz). The two other discs were embedded in biocompatible resin, leaving the cartilage endplate free to permit nutrient diffusion, and fitted in the traction holder; one of these discs was kept in free swelling conditions, whereas the second was submitted to cyclic traction loading (2h/day, 0.2Hz) corresponding to 30% of the animal body weight corrected for organ culture. Results. The cell viability assessed on lactate dehydrogenase and ethidium homodimer stained histological slides was not different between the three cultured discs. This means that the disc viability was not affected neither by the embedding, nor by the traction itself. Compared to the physiologically loaded disc, the gene expression of COL1, COL2 and ACAN was higher in the nucleus pulposus and inner annulus fibrosus of the traction treated disc. In the outer annulus fibrosus of this disc TAGLN and MKX were higher expressed upon traction than in the physiologically loaded disc. Conclusions. Based on these preliminary data, we can conclude that large animal organ culture allows effective unloading of the disc, while preserving cell viability and modulating cellular gene expression responses. This sets the ground for future experiments and opens the door to an evidence-based improvement of clinical spine traction protocols and LBP management overall

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development. Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA. Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA

Few studies have investigated the direct effect of bacteria and their products on articular cartilage chondrocytes ex vivo. An ex vivo model that allows the analysis of chondrocytes in situ would therefore be an important and exciting area of future research. It was hypothesised that a bovine cartilage explant model of septic arthritis would be an ideal model for providing fundamental information on the basic cellular mechanisms of cartilage destruction and chondrocyte death induced by bacterial infection uncomplicated by the immune response. A fresh metacarpophalangeal joint from an abattoir slaughtered 3-year-old cow was skinned, rinsed in water and opened under sterile conditions. The cartilage explants were harvested using surgical scalpels and placed into a total of three tissue culture bottles (2 explants per bottle) containing 10ml Dulbecco's Modified Eagle Medium (DMEM). 50ml of a knee aspirate from a patient with septic arthritis, containing Group B streptococci (GBS), was added to bottle 1, 50ml of a negative knee aspirate was added to bottle 2 and 50ml DMEM to bottle 3. The explants were incubated at 37°C for 24 hours. They were then stained with the fluorescent probes Chloromethylfluorescein Di-acetate (CMFDA) and Propidium Iodide and analysed using a Confocal Scanning Laser Microscope. Cell counts to assess percentage cell death were performed using Velocity 4 software. There was strikingly more cell death observed at 24 hours in the cartilage explant exposed to bacteria in comparison to the non-infected controls. The percentage chondrocyte death was 43% in the presence of GBS, 0.8% in the presence of the negative aspirate and 0.2% in the presence of the DMEM control. Although this is a very preliminary pilot study, it demonstrates an extremely rapid effect on the cartilage. Future bovine explant studies of septic arthritis will therefore be feasible and achievable

Introduction. Sclerostin has been implicated in mechanotransduction in bone and recent data show a lack of response to loading in the sclerostin transgenic mouse. Sclerostin, the protein product of the SOST gene, is an attractive therapeutic target for low bone mass conditions, including osteoporosis. It is expressed exclusively by mature osteocytes in bone and we have shown that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralization and osteoclast activity, as well as inducing catabolic gene expression in osteocytes themselves and promoting osteocyte-mediated bone loss (osteocytic osteolysis). The aim of this study was to examine the direct effects of sclerostin on anabolic responses to loading in bone ex vivo. Methods. 10 × 5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 μstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a second-generation Zetos™ bone loading system. Daily measurements of bone stiffness (Young's modulus), media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made of resin-embedded, non-decalcified samples at the end of the experiment. Gene expression in the bovine bone was examined by real-time RT-PCR. Results. Bovine bone cores showed a steady increase in Young's modulus with daily application of mechanical loading. This increase in stiffness was blocked by the co-addition of sclerostin. Sclerostin also induced bone acidification and a net release of bone calcium, indicated by the decrease in media pH and the relative increase in ionic calcium concentrations in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake. Sclerostin induced an increase in the expression of the bone resorption genes, tartrate resistant acid phosphatase (TRAP), carbonic anhydrase and cathepsin K and induced the release of β-CTX. Histological examination revealed a significant increase in the size of the osteocyte lacunae in sclerostin-treated bone cores, suggesting a role for osteocytic osteolysis in this effect. Discussion/Conclusion. The observation that sclerostin abrogated the loading-induced increase in bone stiffness constitutes direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. Our findings may be explained in part by the observation that sclerostin negatively controls mineralization by late osteoblasts and pre-osteocytes (1). It is also possible that osteocytes themselves are capable of releasing bone mineral in response to sclerostin. This study demonstrates that sclerostin directly antagonises the anabolic effects of mechanical loading in the absence of external (circulating, neural, hormonal) influences. The mechanisms, by which sclerostin exerts these effects, warrant further study

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic. Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation

Formation of micro-cracks occurs in bone due to daily activities. Through a mechanism of self-repair, these micro-cracks are detected, and the damaged areas are restored, avoiding further propagation. The Scissors Model suggests that the osteocyte processes that cross the micro-cracks break as consequence of the cyclic displacements of the micro-crack faces, due to fatigue, and this triggers the remodelling processes. A fresh bovine tibia bone was cut in sections oriented 20° from the transversal direction. The cortical bone was sliced using a circular saw and shaped to the dimensions: 20 × 10 × 1 (mm) and the surfaces were polished. µCT images were obtained from all the samples (μCT 40, Scanco Medical, Brüttisellen, Switzerland). From the DICOM files, the geometries were reconstructed and meshed using tetrahedrons, in ICEM CFD. The Elasticity Modulus (E) was determined in Bonemat, by applying an empirical relationship Elasticity-Density from the literature. The parts were then imported into ANSYS APDL to simulate micro-crack propagation in bone. This model will be validated with further experimental work where the micro-crack will be initiated in the prepared samples and propagated due to fatigue loading, and the osteocyte processes will be visualized in the Scanning Electron Microscope (SEM). This investigation aims to study how cyclic loading in bones and failure of osteocyte processes can trigger target the mechanism of bone remodelling. The resulting model can later contribute for the investigation of treatments for bone diseases such as osteoporosis and the response of bone to the presence of orthopaedic implants

Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine cartilage explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and cartilage explants harvested. Explants were placed into flasks containing Dulbecco's Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability (<1% cell death) over 40hrs incubation with the negative aspirate. However, for the aspirate from a patient positive for S. aureus, there was a rapid increase in cell death between 18 and 24hrs (0.2 +/− 0.3% to 23 +/− 5% cell death respectively) and almost complete cell death at 40hrs (80 +/− 12%; data are means +/− s.d; n=4). These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine cartilage explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, cartilage integrity

Most cases of tendinopathy are believed to be overuse injuries rather than the result of a chronic event. The investigation of the fatigue properties of tendon is therefore of critical importance. This work considered the cyclic stress-relaxation and creep behaviour of two contrasting bovine tendon types – the largely postional digital extensor and the more energy storing deep digital flexor tendon. Fascicles were cyclically loaded (1Hz), to 1800 cycles of stress relaxation or to failure in creep, stopping some tests at 300, 900 or 1200 cycles to perform quasi-static failure tests or confocal imaging using a highly concentrated Acridine Orange solution. Creep tests were cycled to 60% of the ultimate tensile strength (UTS), while for stress relaxation, cyclic deformation to the strain associated with 60% UTS was used. Flexor tendon fascicles were found to exhibit reduced stress relaxation at all time points compared to the extensor fascicles and also showed an increase in the mean cycles to failure during creep testing. Evidence of fatigue damage was clear in the confocal images with breakdown of the collagen fibre alignment evident from 300 cycles; however it appears that some damage could occur without effect on the UTS of the fascicle. Despite what appears to be superior fatigue resistance in the flexor tendon fascicles, the matrix damage, certainly at early time points, appeared visually to be as severe as that observed with the extensor tendon fascicles

Objective. To study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro. Methods. Chondrocytes were harvested from bovine femoral condyle cartilage and isolated using collagenase-containing media. At 24 hours after seeding 15 000 cells per well onto a 96-well plate, chondrocytes were treated with media (DMEM/F12 + ITS), PBS, 1:1 lidocaine (2%):PBS, 1:1 bupivacaine (0.5%):PBS, 1:1 lidocaine (2%):HA, 1:1 bupivacaine (0. 5%):HA, or 1:1 HA:PBS for one hour. Following treatment, groups had conditions removed and 24-hour incubation. Cell viability was assessed using PrestoBlue and confirmed visually using fluorescence microscopy. Results. Media-treated groups had a mean of 1.55×10. 4. cells/well (. sem. 783). All treated cells showed statistically significant reduced viability when compared with media alone (all p < 0.003). Cells treated with bupivacaine + HA (6.70×10. 3. cells/well (. sem. 1.10×10. 3. )) survived significantly more than bupivacaine (2.44×10. 3. cells/well (. sem . 830)) (p < 0.001). Lidocaine + HA (1.45×10. 3. cells/well (. sem. 596)) was not significantly more cytotoxic than lidocaine (2.24×10. 3. cells/well (. sem. 341)) (p = 0.999). There was no statistical difference between the chondrotoxicities of PBS (8.49×10. 3. cells/well (. sem. 730) cells/well) and HA (4.75×10. 3. cells/well (. sem. 886)) (p = 0.294). Conclusions. HA co-administration reduced anaesthetic cytotoxicity with bupivacaine but not lidocaine, suggesting different mechanisms of injury between the two. Co-administered intra-articular injections of HA with bupivacaine, but not lidocaine, may protect articular chondrocytes from local anaesthetic-associated death. Cite this article: Bone Joint Res 2013;2:270–5

Intra-articular screw fixation is indicated for internal fixation of large osteochondral fragments secondary to trauma or osteochondritis dissecans. During surgery, orthopaedic drills are used to prepare a hole through which the screw can pass. Previous work has shown that mechanical injury to articular cartilage results in a zone of cell death adjacent to the traumatised articular cartilage (1). Here, we characterise and quantify the margin of in situ chondrocyte death surrounding drill holes and screws (standard cortical and headless compression designs) placed in mature bovine articular cartilage to model the orthopaedic procedure. Drill holes (1mm) were made through the articular cartilage and bone of intact bovine metacarpophalangeal joints obtained from 3-yr old cows within 12hrs of slaughter. Osteochondral explants (∼1cm square and 2-3mm thick) encompassing the drilled holes in articular cartilage and subchondral bone were harvested using a chisel. Explants were then incubated in Dulbecco's modified Eagle's medium for 45mins with CMFDA (5-chloromethylfluorescein diacetate) and PI (propidium iodide; both at 10micromolar) to identify/quantify living and dead in situ chondrocytes respectively in a consecutive series of axial optical sections using confocal scanning laser microscopy (CLSM). The drill holes through cartilage appeared to have clearly defined edges with no macroscopic evidence of cartilage splitting. However visualisation of fluorescently-labelled in situ chondrocytes by CLSM demonstrated clear cell death around the periphery of the drilled hole which was 166±19 micrometers in width. This increased with a larger diameter (1.5mm) drill to 450±151 micrometers (all data are means±s.e.m.; n=3). Preliminary experiments indicated that the margin of chondrocyte death around a 1.5mm hole was dramatically increased further by the insertion of screws into pre-drilled holes. These results suggest that the mechanical trauma associated with cartilage drilling and the insertion of intra-articular screws occurs with marked death of in situ chondrocytes extending into normal cartilage beyond the area occupied by the screw. As chondrocytes are not replaced in mature cartilage, their loss around the hole/screw will mean that the extracellular matrix is not maintained, inevitably leading to cartilage failure

Introduction. Low back pain is a major public health problem in our society. Degeneration of intervertebral disc (IVD) appears to be the leading cause of chronic low-back pain [1]. Mechanical stimulations including compressive and tensional forces are directly implicated in IVD degeneration. Several studies have implicated the cytoskeleton in mechanotransduction [2, 3], which is important for communication and transport between the cells and extracellular matrix (ECM). However, the potential roles of the cytoskeletal elements in the mechanotransduction pathways in IVD are largely unknown. Methods. Outer annulus fibrosus (OAF) and nucleus pulposus (NP) cells from skeletally mature bovine IVD were either seeded onto Flexcell¯ type I collagen coated plates or seeded in 3% agarose gels, respectively. OAF cells were subjected to cyclic tensile strain (10%, 1Hz) and NP cells to cyclic compressive strain (10%, 1Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy and RNA extracted for quantitative PCR analysis. Results. F-actin reorganisation was evident in OAF and NP cells subjected to tensile and compressive strain respectively and is likely due to load-induced differential mRNA expression of actin-binding proteins. The vimentin network was also more intricately organised in loaded NP cells. Compressive strain increased type II collagen and aggrecan transcription in NP cells, whereas levels decreased in OAF cells under tension. mRNA levels of ECM-degrading enzymes were significantly reduced in both cell populations after loading. Conclusion. Tensile and compressive strains induce different mechano-responses in the organisation/expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ

Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p<0.001) compared to the alpha-toxin knockout strain, which was negligible (4.1+/−1.7%; means+/−SEM; N=4 independent experiments). In this in vitro bovine cartilage explant model, whereby the effects of defined toxins were determined in isolation of a complex host immune response, in situ chondrocyte viability was dramatically and exclusively reduced by alpha-toxin. This work forms the basis for developing a rational treatment to reduce the extent of cartilage destruction during an episode of septic arthritis. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints. Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation. A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment. We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift

Mechanical loading is important to maintain the homeostasis of the intervertebral disc (IVD) under physiological conditions but can also accelerate cell death and tissue breakdown in a degenerative state. Bioreactor loaded whole organ cultures are instrumental for investigating the effects of the mechanical environment on the IVD integrity and for preclinical testing of new therapies under simulated physiological conditions. Thereby the loading parameters that determine the beneficial or detrimental reactions largely depend on the IVD model and its preparation. Within this symposium we are discussing the use of bovine caudal IVD culture models to reproduce tissue inflammation or matrix degradation with or without bioreactor controlled mechanical loading. Furthermore, the outcome parameters that define the degenerative state of the whole IVD model will be outlined. Besides the disc height, matrix integrity, cell viability and phenotype expression, the tissue secretome can provide indications about potential interactions of the IVD with other cell types such as neurons. Finally, a novel multiaxial bioreactor setup capable of mimicking the six degrees-of-freedom loading environment of IVDs will be introduced that further advances the relevance of preclinical ex-vivo testing

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering