Total ankle replacement (TAR) is the main surgical option in case of severe joint osteoarthritis. The high failure rate of current TAR is often associated to inappropriate prosthetic articulating surfaces designed according to old biomechanical concepts such the fixed axis of rotation, thus resulting in non-physiological joint motion. A recent image-based 3D morphological study of the normal ankle (Siegler et al. 2014) has demonstrated that the ankle joint surfaces can be approximated by a saddle-shaped cone with its apex located laterally (SSCL). We aimed at comparing the kinematic effects of this original solution both with the intact joint and with the traditional prosthetic articulating surfaces via in-silico models and in-vitro measurements. Native 3D morphology of ten normal cadaver ankle specimens was reconstructed via MRI and CT images. Three custom-fit ankle joint models were then developed, according to the most common TAR designs: cylindrical, symmetrically-truncated medial apex cone (as in Inman's pioneering measures), and the novel lateral apex cone, i.e. SSCL. Bone-to-bone motion, surface-to-surface distance maps, and ligament forces and deformations were evaluated via computer simulation. Prototypes of corresponding prosthesis components were designed and manufactured via 3D-printing, both in polymer-like-carbon and in cobalt-chromium-molybdenum powders, for in-vitro tests on the cadaver specimens. A custom testing rig was used for application of external moments to the ankle joint in the three anatomical planes; a motion tracking system with trackers pinned into the bone was used to measure tibial, talar and calcaneal motion (Franci et al. 2009), represented then as tibiotalar, subtalar and ankle complex 3D joint rotations. Each ankle specimen was tested in the intact joint configuration and after replacement of the articulating surfaces according with the three joint models: cylindrical, medial apex cone and SSCL. Results. Small intra-specimen data variability in cycle-to-cycle joint kinematics was found in all cadaver ankles, the maximum standard deviation of all rotation patterns being smaller than 2.0 deg. In-silico ligament strain/stress analysis and in-vitro joint kinematic and load transfer measurements revealed that the novel SSCL surfaces reproduce more natural joint patterns than those with the most common surfaces used in current TAR. TAR based on a saddle-shaped skewed truncated cone with lateral apex is expected to restore more normal joint function. Additional tests are undergoing for further biomechanical validation. The present study has also demonstrated the feasibility and the quality of the full process of custom TAR design and production for any specific subject. This implies a thorough procedure, from medical imaging to the production of artificial surfaces via 3D printing, which is allowing for personalised implants to become the future standard in total joint replacement

Introduction. The National Joint Registry has recently identified failure of large head metal on metal hip replacements. This failure is associated with the high torque at the interface of standard modular taper junction leading to fretting and corrosion. A number of manufacturers produce mini spigots, which in theory, provide a greater range of motion as the neck head junction is reduced. However, the relative torque to interface ratio at this junction is also increased. In this study we investigated hypothesis that the use of small spigots (minispigots) will increase wear and corrosion on modular tapers. Methods. Wear and corrosion of spigots were compared in-vitro when loaded with a force representative of the resultant force passing through the hip. The heads (female tapers) were made of cobalt-chrome-molybdenum (CoCrMo) and the stems (male tapers) of titanium alloy (Ti). Commercially available tapers and heads were used. The surface parameters & profiles were measured before & after testing. Electrochemical static and dynamic corrosion (pitting) tests were performed on minispigots under loaded and non-loaded conditions. Results. Post-testing the surface parameters Ra, Ry & Rz on the head taper associated with the minispigots had become greater compared with standard spigots. In all instances the profile of the titanium male tapers was unchanged. SEM showed the corroded region of the head was similar to the profile on the Ti male taper, with evidence of pitting in the cobalt chrome. In the CoCrMo/ Ti combinations, wear and corrosion were increased in minispigots compared with standard spigots. On minispigots the rough surface finishes were affected more severely than those with a smoother surface. Static corrosion tests showed evidence of fretting in the rough but not the smooth minispigots. Pitting scans showed a greater hysteresis with the rough surface finishes on the minispogot indicating potentially greater corrosion in the former. Conclusion. The relative size of the taper in comparison to the head combined with the surface finish was crucial. As the relative torque to interface ratio at this junction increased corrosion of the cobalt chrome head increases and is further enhanced if the surface finish on the tapers is rough

The heat produced by drills, saws and PMMA cement in the handling of bone can cause thermal necrosis. Thermal necrosis could be a factor in the formation of a fibrous tissue membrane and impaired bony ingrowth into porous prostheses. This has been proposed to lead to non-union of osteotomies and fractures, the failure of the bone-cement interface and the failure of resurfacing arthroplasty. We compared three proprietary blades (the De Soutter, the Stryker Dual Cut and the Stryker Precision) in an in-vitro setting with porcine tibiae, using thermocouples embedded in the bone below the cutting surface to measure the increases in bone temperature. There was a significant (p=0.001) difference in the change in temperature (δT) between the blade types. The mean increase in temperature was highest for the De Soutter, 2.84°C (SD: 1.83°C, range 0.48°C to 9.30°C); mean δT was 1.81°C (SD: 1.00°C, range 0.18°C to 4.85°C) for the Precision and 1.68°C (SD: 0.95°C, range 0.24°C to 5.67°C). Performing paired tests, there was no significant difference in δT between the Precision and Dual Cut blades (p=0.340), but both these blades had significantly (p=0.003 for Precision vs De Soutter, p<0.001 for Dual Cut vs De Soutter) lower values for δT than the Dual Cut

Tribology and wear of articular cartilage is associated with the mechanical properties, which are governed by the extracellular matrix (ECM). The ECM adapts to resist the loads and motions applied to the tissue. Most investigations take cartilage samples from quadrupeds, where the loading and motions are different to human. However, very few studies have investigated the differences between human and animal femoral head geometry and the mechanical properties of cartilage.

This study assessed the differences between human, porcine, ovine and bovine cartilage from the femoral head; in terms of anatomical geometry, thickness, equilibrium elastic modulus and permeability.

Diameter of porcine (3-6 months old), bovine (18-24 months old), ovine (4 years old) and human femoral heads were measured (n=6). Plugs taken out of the superior region of each femoral head and creep indentation was performed. The human femoral heads were obtained from surgery due to femoral neck fracture. Cartilage thickness was measured by monitoring the resistive force change as a needle traversed the cartilage and bone at a constant feed rate using a mechanical testing machine. The percentage deformation over time was determined by dividing deformation by thickness. A biphasic finite element model was used to obtain the intrinsic material properties of each plug. Data is presented as the mean ± 95% confidence limits. One-way ANOVA was used to test for significant differences (p < or = 0.05).

Significant differences in average femoral head diameter were observed between all animals, where bovine showed the largest femoral head. Human cartilage was found to be significantly thicker than cartilage from all quadrupedal hips. Human cartilage had a significantly larger equilibrium elastic modulus compared to porcine and bovine cartilage. Porcine articular cartilage was measured to be the most permeable which was significantly larger than all the other species. No significant difference in permeability was observed between human and the other two animals: bovine and ovine (Table 1).

The current study has shown that articular cartilage mechanical properties, thickness and geometry of the femoral heads differ significantly between different species. Therefore, it is necessary to consider these variations when choosing animal tissue to represent human.

In-vitro models of disease are valuable tools for studying disease and analysing response to therapeutics. Recently, advances in patient-derived organoid (PDO) models have been shown to faithfully recapitulate structure, function, and therapeutic response for a wide range of tissues. Frozen shoulder is a rare example of a chronic inflammatory fibrotic disease which is self-limiting, unlike many other soft tissue fibrotic disorders. As no in-vitro 3D models or in-vivo animal models exist for frozen shoulder, establishing an organoid model which recapitulates core diseases features may give insight into fibrosis resolution. Consequently, using biocompatible hydrogels, primary capsular fibroblasts, monocyte-derived macrophages and HUVEC cells, we generated stable PDO cultures which exhibited key disease phenotypes, including vascularization, increased stiffness, and an expanded lining layer over 21 days of culture. Through further investigation of cell-matrix and cell-cell interactions in the organoid model, we intend to unpack the differences between resolving and non-resolving fibrotic disease and uncover clinically relevant therapeutic targets for fibrosis

The most common reason for revision surgery of total hip replacements is aseptic loosening of implants secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can cause pseudotumour formation. As revision surgery is associated with higher mortality and infection, it is important to understand the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4) has been shown to mediate immune responses to cobalt ions. Statin use in epidemiological studies has been associated with reduced risk of revision surgery. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses and there is evidence that statins can modulate TLR4 activity. This study investigates simvastatin's effect on orthopaedic biomaterial-mediated changes in protein expression of key inflammatory markers and soluble-ICAM-1 (sICAM-1), an angiogenic factor implicated in pseudotumour formation. Human macrophage THP-1 cells were pre-incubated with 50µM simvastatin for 2-hours or a vehicle control (VC), before being exposed to 0.75mM cobalt chloride, 50μm3 per cell zirconium oxide or LPS as a positive control, in addition to a further 24-hour co-incubation with 50µM simvastatin or VC. Interleukin −8 (IL-8), sICAM-1, chemokine ligand 2 (CCL2), CCL3 and CCL4 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). GraphPad Prism 10 was used for statistical analysis including a one-way ANOVA. Pre-treatment with simvastatin significantly reduced LPS and cobalt-mediated IL-8 secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells. Pre-treatment with simvastatin significantly reduced LPS-mediated but not cobalt ion-mediated CCL2 (n=3) and CCL3 protein (n=3) secretion in THP-1 cells. Simvastatin significantly reduced zirconium oxide-mediated CCL4 secretion (n=3). Simvastatin significantly reduced cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving implant failure

Abstract. Objectives. The enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production. Methods. Cell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×10. 4. , 5×10. 4. , 1×10. 5. , 2×10. 5. in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope. 3D mini-tissue constructs. 15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix. Results. RTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×10. 5. RTFs and 3×10. 5. BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage. Conclusions. This study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Abstract. OBJECTIVES. Dual mobility (DM) total hip replacements (THRs) were introduced to reduce the risk of hip dislocation in at-risk patients. DM THRs have shown good overall survivorship and low rates of dislocation, however, the mechanisms which describe how these bearings function in-vivo are not fully understood. This is partly due to a lack of suitable characterisation methodologies which are appropriate for the novel geometry and function of DM polyethylene liners, whereby both surfaces are subject to articulation. This study aimed to develop a novel semi-quantitative geometric characterisation methodology to assess the wear/deformation of DM liners. METHODS. Three-dimensional coordinate data of the internal and external surfaces of 14 in-vitro tested DM liners was collected using a Legex 322 coordinate measuring machine. Data was input into a custom Matlab script, whereby the unworn reference geometry was determined using a sphere fitting algorithm. The analysis method determined the geometric variance of each point from the reference surface and produced surface deviation heatmaps to visualise areas of wear/deformation. Repeatability of the method was also assessed. RESULTS. Semi-quantitative analysis of the surface deviation heatmaps revealed circumferential damage patterns similar to those reported in the literature. Additionally, the location of the damaged regions corresponded between the internal and external surfaces. Comparing five repeat measurements of the same liner, the maximum geometric variance of each surface varied by 1 µm (standard deviation) suggesting a high repeatability of the method. CONCLUSIONS. This study presents an effective and highly repeatable characterisation methodology to semi-quantitatively assess the wear/deformation of in-vitro tested DM liners. This method is suitable for the analysis of retrieved DM liners whereby no pre-service information is available, which may provide information about the complex in-vivo kinematics and mechanical failure mechanisms of these bearings

Production of porous titanium bone implants is a highly promising research and application area due to providing high osseointegration and achieving the desired mechanical properties. Production of controlled porosity in titanium implants is possible with laser powder bed fusion (L- PBF) technology. The main topics of this presentation includes the L-PBF process parameter optimization to manufacture thin walls of porous titanium structures with almost full density and good mechanical properties as well as good dimensional accuracy. Moreover, the cleaning and coating process of these structures to further increase osseointegration and then in-vitro biocompatibility will be covered

Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Pre-treatment with simvastatin significantly reduced cobalt-mediated IL-8 protein secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells (p-value<0.0001). Work will be undertaken to determine changes in gene expression, the role of TLR4 in these responses and the effect of simvastatin on additional inflammatory markers. Conclusions. Simvastatin significantly reduces cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving aseptic loosening and pseudotumour formation

To design slow resorption patient-specific bone graft whose properties of bone regeneration are increased by its geometry and composition and to assess it in in-vitro and in-vivo models. A graft composed by hydroxyapatite (HA) and β-TCP was designed as a cylinder with 3D gyroid porosities and 7 mm medullary space based on swine's anatomy. It was produced using a stereolithography 3D-printing machine (V6000, Prodways). Sterile bone grafts impregnated with or without a 10µg/mL porcine BMP-2 (pBMP-2) solution were implanted into porcine femurs in a bone loss model. Bone defect was bi-weekly evaluated by X-ray during 3 months. After sacrifice, microscanner and non-decalcified histology analysis were conducted on biopsies. Finally, osteoblasts were cultured inside the bone graft or in monolayer underneath the bone graft. Cell viability, proliferation, and gene expression were assessed after 7 and 14 days of cell culture (n=3 patients). 3D scaffolds were successfully manufactured with a composition of 80% HA and 20% β-TCP ±5% with indentation compressive strength of 4.14 MPa and bending strength of 11.8MPa. In vivo study showed that bone regeneration was highly improved in presence of pBMP-2. Micro-CT shows a filling of the gyroid sinuses of the implant (Figure 1). In vitro, the presence of BMP2 did not influence the viability of the osteoblasts and the mortality remained below 3%. After 7 days, the presence of BMP2 in the scaffold significantly increased by 85 and 65% the COL1A1 expression and by 8 and 33-fold the TNAP expression by osteoblasts in the monolayer or in the scaffold, respectively. This BMP2 effect was transient in monolayer and did not modify gene expression at day 14. BMP2-impregnated bone graft is a promising patient-personalized 3D-printed solution for bone defect regeneration, by promoting neighboring host cells recruitment and solid new bone formation. For any figures and tables, please contact the authors directly

Osteosynthesis aims to maintain fracture reduction until bone healing occurs, which is not achieved in case of mechanical fixation failure. One form of failure is plastic plate bending due to overloading, occurring in up to 17% of midshaft fracture cases and often necessitating reoperation. This study aimed to replicate in-vivo conditions in a cadaveric experiment and to validate a finite element (FE) simulation to predict plastic plate bending. Six cadaveric bones were used to replicate an established ovine tibial osteotomy model with locking plates in-vitro with two implant materials (titanium, steel) and three fracture gap sizes (30, 60, 80 mm). The constructs were tested monotonically until plastic plate deformation under axial compression. Specimen-specific FE models were created from CT images. Implant material properties were determined using uniaxial tensile testing of dog bone shaped samples. The experimental tests were replicated in the simulations. Stiffness, yield, and maximum loads were compared between the experiment and FE models. Implant material properties (Young's modulus and yield stress) for steel and titanium were 184 GPa and 875 MPa, and 105 GPa and 761 MPa, respectively. Yield and maximum loads of constructs ranged between 469–491 N and 652–683 N, and 759–995 N and 1252–1600 N for steel and titanium fixations, respectively. FE models accurately and quantitatively correctly predicted experimental results for stiffness (R2=0.96), yield (R2=0.97), and ultimate load (R2=0.97). FE simulations accurately predicted plastic plate bending in osteosynthesis constructs. Construct behavior was predominantly driven by the implant itself, highlighting the importance of modelling correct material properties of metal. The validated FE models could predict subject-specific load bearing capacity of osteosyntheses in vivo in preclinical or clinical studies. Acknowledgements: This study was supported by the AO Foundation via the AOTRAUMA Network (Grant No.: AR2021_03)

Entheses are the anchorage sites of tendons to bones in the musculoskeletal system. They have a unique microanatomy that allow smooth transfer of mechanical load through tendon to bone. However, entheses are prone to injury due to their small surface area. 1,2. The overall success rate of the current gold standard treatment (directly attaching the tendon to bone) is small. 3,4. Consequently, the aim of this study was to evaluate different hydrogels and their suitability for developing an in-vitro co-culture system to manufacture 3D tissue interfaces. To create a 3D in-vitro tissue interface, half-well plugs were created by pouring silicone in wells of a 24-well plate. When set, it was cut into halves to be used as half-well plugs, blocking one side of a culture well. A tendon-cell-encapsulated hydrogel was poured into the exposed half and, when set, the plug was removed and a bone-cell-encapsulated gel was added. Cells were fluorescently labelled to enable identification of cell types under fluorescent microscopy (Tendon – green, bone – red). The suitability of different hydrogels to form an in vitro tissue interface was evaluated: fibrin, agarose and gellan. This study demonstrates that 3D co-cultures can be manufactured in-vitro. The novel system enabled the culture of two cell types (bone/tendon) in direct contact, creating an in-vitro interface. In addition, this study shows that fibrin gel supports cell morphology, while both cell types failed to show normal morphology in agarose and gellan. Further studies evaluating cell viability in these hydrogels are currently underway

Worldwide, osteoporosis, causes more than 8.9 million fractures annually, resulting in an osteoporotic fracture every 3 seconds, where 1 in every 3 women and 1 in every 5 men aged over 50 will experience osteoporotic fractures at least once in their lifetime. Vertebral fractures, estimated at 1.4 million/year are among the most common fractures, posing enormous health and socioeconomic challenges to the individual and society at large. Considering that the great majority of individuals at high risk (up to 80%), who have already had at least one osteoporotic fracture, are neither identified nor treated, prediction of the risk factors for vertebral fractures can be of great value for prevention/early diagnosis. Recent studies show that finite element analysis of computed tomography (CT) scans provides noninvasive means to assess fracture risk and has the potential to be clinically implemented upon proper validation. The objective of this study was to develop a voxel-based finite element model using quantitative computed tomography (QCT) images in conjunction with in-vitro experiments to evaluate the strength of the vertebral bodies and predict the fracture risk criteria. A total of 10 vertebrae were dissected from juvenile sheep lumbar spines. The attached soft tissues and posterior elements and facet joints were completely removed, and the upper and lower vertebral bodies were polished using glass paper to provide smooth surfaces. The specimens were wrapped in phosphate buffer saline (PBS) soaked gauze, sealed in plastic bags, and stored in a refrigerator at −22°C. QCT scans of the specimens were captured using a bone density calibration phantom (QRM Co., Moehrendorf, Germany) with three 18 mm cylindrical inserts, providing 0, 100 and 200 mg HA/ccm, respectively. All the specimens, preserved hydrated in PBS solution, were mechanically tested at room temperature using a mechanical testing apparatus (Zwick/Roell, Ulm-Germany). The QCT images were then used to reconstruct the voxel-based FE model employing a custom-developed heterogeneous material mapping code. Five different equations for the correlation of the density and the elastic modulus were used to validate the efficiency of the FE model as compared to the in-vitro experiments. The results of the voxel-based FE models matched well with the in-vitro experiments, with an average error of 11.38 (±4.09)% based on the power law equation. A failure criterion was embedded in the FE models and the initiation of fracture was successfully predicted for all specimens. Further, typical kyphoplasty treatment was simulated in the 5 models to evaluate the application of the validated algorithm in the estimation of the failure patterns. Our novel voxel-based FE model can be used in future studies to predict the outcome of different types of therapeutic modalities/surgeries and estimate fracture risk including postoperative fractures

Low back pain (LBP) is the leading cause of disability worldwide, interfering with an individual's quality of life and work performance. Understanding the degeneration mechanism of the intervertebral disc (IVD), one of the key triggers of LBP, is hence of great interest. Disc degeneration can be mimicked in animal studies using the injection of enzymatic digestion, needle puncture, stab injury, or mechanical over-loading [1]. However, the detailed response of the artificial degenerated disc using needle puncture under physiological dynamic loading in diurnal activities has not yet been analyzed using FE-models. To fill the gap in literature, this study investigates the role of needle puncture injury on the biomechanical response of IVD using a combination of Finite Element (FE) simulations and in-vitro lumbar spine sheep experiments. 16 lumbar motion segments (LMS) were dissected from juvenile sheep lumbar spines. The harvested LMSs were assigned equally to two groups (control group with no incision and an injured group punctured with a 16-gauge needle). All specimens were mounted in a homemade chamber filled with saline solution and underwent a stress-relaxation test using a mechanical testing apparatus (Zwick/Roell, Ulm-Germany). A validated inverse poroelastic FE methodology [2] in conjunction with in-vitro experiments were used to find the elastic modulus and permeability. Subsequently, specimen-specific FE models for the 16 discs were simulated based on daily dynamic physiological activity (i.e., 8h rest followed by a 16h loading phase under compressive loads of 350 N and 1000 N, respectively). The results of the individual FE models were well fitted with the in-vitro stress-relaxation experiments, with an average error of 7.48 (±2.24)%. The results of the simulations demonstrated that the variation of axial displacement in the control discs was significantly higher than the injured ones (P=0.037). At the end of day, the intradiscal pressure (IDP) was slightly higher in the control group (P=0.061) although the maximum axial stress in the annulus fibrosus (AF) was significantly higher in the injured group (P=0.028). The total fluid loss after 24h was significantly higher in the control group (p<0.001). We found that needle puncture can decrease the strain range, IDP, and fluid loss in an IVD, although it increases the axial stress. We therefore hypothesize that the fissures, clefts or tears produced by needle puncture alter the saturation time for disc deformation and pore pressure. The collapsed disc structure hinders the fluid flow capability; hence, the total fluid loss decreases for the injured discs, inhibiting the transportation of nutrients. Higher stresses in the AF were observed for the injured group in alignment with previous studies [3]. It is therefore concluded that the needle puncture injury methodology can be effectively used to mimic the degeneration mechanism in animal models. It is a convenient, reproducible, and cost-effective technique. Future work includes exploring degenerated disks induced by needle puncture to investigate potential regenerative therapeutics

Abstract. Introduction. Altered mechanical loading is a contributing factor to low back pain, a condition affecting 80% of the population at some point in life. A plethora of in-vitro studies exist focusing on 6 degree of freedom (dof) testing of functional spinal units (FSU) to obtain a specimen stiffness matrix. Due to differences in the performance of test apparatus and in the technique used to manipulate raw data it is difficult to compare results from different groups. Objectives. Our primary objective was to develop a standardised technique to benchmark the performance of testing apparatus; a secondary objective was to standardise the data manipulation technique. Methods. 6 tests each at 250N and 500N preload were performed on synthetic FSU specimens using the Bath spine simulator, with a further 3 tests performed on porcine specimens. Three techniques were used to evaluate stiffness: first the slope of the entire load-displacement curve, inclusive of loading and unloading portions, was considered, secondly zonal stiffnesses were defined by dividing the load displacement curve into elastic and neutral zones, finally stiffness was calculated only for the loading portion of the elastic and neutral zone. The standard error of the residuals was used to compare results. Results. The stiffness matrix principal elements of the synthetic FSU showed repeatability of 3.2% and reflected specimen symmetry in the x and y (8% error). The stiffness calculation techniques including both loading and unloading portions of the curve were affected by hysteresis, an issue that did not arise when only the loading portion was used in calculations. Conclusions. It is recommended that testing apparatus performance is evaluated with synthetic specimens, to allow benchmarking against different set-ups. Furthermore, it is recommended that stiffness calculations are performed only on the loading part of the curve to eliminate the influence of hysteresis on the results. 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

Background. In-vitro testing of knee joints remains vital in the understanding of knee surgery and arthroplasty. However, based on the design philosophy of the original Oxford knee simulator, this in-vitro testing has mainly focused on squatting motion. As the activities of daily living might drastically differ from this type of motion, both from a kinematic and kinetic point of view, a new knee simulator is required that allows studying more random motion patterns. This paper describes a novel knee simulator that overcomes the limitations of traditional Oxford simulators, providing both kinematic and kinetic freedom with respect to the applied boundary conditions. Methods. This novel test simulator keeps the hip at a fixed position, only providing a single rotational degree of freedom (DOF) in the sagittal plane. In addition, the ankle holds four DOF, including all rotational DOF and the translation along the medio-lateral axis. Combining these boundary conditions leaves five independent DOF to the knee; the knee flexion angle is actively controlled through the positioning of the ankle joint in the antero-posterior and proximal-distal direction. The specimens' quadriceps muscle is actively controlled, the medial and lateral hamstrings are passively loaded. To validate the performance of this simulator, two fresh frozen specimens have been tested during normal squatting and cycling. Their kinematic patterns have been compared to relevant literature data. Results. Kinematic patterns in line with literature data are observed for the squatting motion, e.g. displaying femoral rollback for both specimens. In contrast, the kinematic patterns that are observed during cycling differ remarkably from the patterns of the squatting movement. Conclusion. The results provide confidence in the working principle of the presented knee simulator, the mechanical design and all processing steps. In addition, the remarkable differences observed in kinematic patterns between different studied motions indicate the need for broadening the research view to relevant motion patterns, beyond squatting

Introduction. Ischaemic preconditioning (IPC) is a phenomenon whereby a tissue is more tolerant to an insult if it is first subjected to short bursts of sublethal ischaemia and reperfusion. The potential of this powerful mechanism has been realised in many branches of medicine where there is an abundance of ongoing research. However, there has been a notable lack of development of the concept in Orthopaedic surgery. The routine use of tourniquet-controlled limb surgery and traumatic soft tissue damage are just two examples of where IPC could be utilised to beneficial effect in Orthopaedic surgery. Methods. We conducted a randomized controlled clinical trial looking at the role of a delayed remote IPC stimulus on a cohort of patients undergoing a total knee arthroplasty (TKA). We measured the effect of IPC by analysing gene expression in skeletal muscle samples from these patients. Specifically we looked at the expression of Heat shock protein-90 (HSP-90), Catalase and Cyclo-oxygenase-2 (COX-2) at the start of surgery and at one hour into surgery. Gene analysis was performed using real time polymerase chain reaction amplification. As a second arm to the project we developed an in-vitro model of IPC using a human skeletal muscle cell line. A model was developed, tested and subsequently used to produce a simulated IPC stimulus prior to a simulated ischaemia-reperfusion (IR) injury. The effect of this on cell viability was investigated using crystal violet staining. Results. In the clinical arm of the study 4 patients were randomized to a control group and 4 randomized to IPC. Operative and post-operative periods were without any adverse incident. For each gene in question there was a different pattern in expression. COX-2 showed an initial up-regulation of 1.43 (p=0.83) at the start of surgery and a subsequent down-regulation of 0.07 (p=0.01) at one hour into surgery. Catalase expression was lower than control at the start of surgery (0.62, p= 0.46) and at one hour into surgery (0.5, p=0.1). HSP-90 expression was initially lower than control at the start of surgery (0.59, p= 0.07) then up-regulated at one hour into surgery (1.13, p=0.62). In the in-vitro section of the study we found that 15 hours of simulated ischaemia was required for a cell death of approximately 50 % (p=0.00001). The introduction of a simulated IPC stimulus increased cell death at a 1 hour reperfusion time-point (IPC group had 18% more cell death than IR group, p=0.003) and at a 24 hour reperfusion time-point (IPC group had 19% more cell death than IR group, p= 0.00001). At a 72 hours reperfusion time-point the IPC group had a 30% greater survival than the IR group (p=0.000006). Conclusion. Our clinical study was subject to small sample size. Despite this it suggests a particular importance of COX-2 in the IPC mechanism. The in-vitro model we developed is an essential resource for further studies into IPC in Orthopaedic Surgery. Preliminary results from this model point towards the ‘second window of protection’ of IPC as a stronger phenomenon than immediate preconditioning

By combining cells, biological factors, and biomaterials the field of tissue engineering has generated technologies capable of supporting regeneration. However, the regulatory hurdles associated with the use of cell-based therapies often hinder translation. Consequently, to meet the growing demand for regenerative technologies new approaches are needed. Emerging evidence suggests that cell-derived extracellular vesicles (EVs) are critical in cell-cell communication and regulation of bone formation. This talk will explore the role of osteoblast EVs in directing stem-cell differentiation in-vitro. EVs were isolated from cell culture media by ultracentrifugation and profiled for size and composition using a range of techniques. Notably, proteomic analysis revealed the presence of calcium channelling annexins and bridging collagens that may be key to their role in mineralisation. To minimise the concentration of EVs required to induce a pro-osteogenic effect we propose that they may be locally delivered. Opportunities to incorporate these pro-osteogenic EVs into injectable biomaterials will be discussed, in particular the formulation of microcapsules and fluid-gels. In summary, incorporation of EVs in tissue-engineered scaffolds has the potential to deliver all the advantages of a cell-based therapy but without using viable cells. The advantages of this approach may represent a new phase of tissue engineering

Intraosseous Transcutaneous Amputation Prosthesis (ITAP) is a new generation of limb replacements that can provide to amputees, an alternative solution to the main problems caused by the most common used external prosthesis such as pressure sores, infections and unnatural gait. ITAP is designed as one pylon osteointegrated into the bone and protruding through the skin, allowing both the mechanical forces to be directly transferred to the skeleton and the external skin being free from frictions and infections. The skin attachment to the implant is fundamental for the success of the ITAP, as it prevents the implant to move and consequently fail. In this study we wanted to test if cell viability and attachment was improved using TiO2 nanotubes. Human keratinocytes and human dermal fibroblasts were seeded for three days on TiO2 nanotubes with different sizes (18–30nm, 40–60nm and 60–110nm), compared with controls (smooth titanium) and tested for viability and attachment. A Mann-Whitney U test was used to compare groups where p values < 0.05 were considered significant. The results showed that the viability and cell attachment for keratinocytes were significantly higher after three days on controls comparing with all nanotubes (p=0.02), while attachment was higher on bigger nanotubes and controls. Cell viability for fibroblasts was significantly higher on nanotubes between 40 and 110nm comparing with smaller size and controls (p=0.03), while investigation of cell attachment is ongoing. From these early results, we can say that TiO2 nanotubes can improve the soft tissue attachment on ITAP. Further in-vitro and ex-vivo experiments on cell attachment will be carried out