Abstract. Introduction. Back pain affects 80% of the population at some stage in their life with significant costs to society. Mechanisms and causes of pain have been investigated by studying the behaviour of functional spinal units (FSUs) subjected to displacement- or load control protocols in 6 degrees of freedom (DOF). Load control allows specimens to move physiologically in response to applied loads whereas displacement control constrains motion to individual axes. The displacement control system of the Bath University six-axis spine simulator has been validated and the load control system is in the process of iterative development. Objectives. The objective was to build a computational model of the spine simulator to develop a complete 6 DOF load control system to enable accurate specimen testing under load control. Methods. SolidEdge part files of the simulator assembly exported to MATLAB Simulink® were used to generate a full model of the simulator. Results from displacement tests using a helical spring specimen in the simulator were used to validate the performance of the simulator model in displacement control. The model was then used to develop a 6 DOF load control system including matrix transformations to ensure correct load tracking. Results. Model results for displacement control matched the physical test data within 12% and replicated coupling loads. The developed load control model demonstrated good control in all 6 axes, maintaining zero-commanded loads. Furthermore, peak-to-peak errors in non-zero-commanded loads and moments were below 10% and 15% respectively. Conclusions. The computational model proved a valuable tool in understanding the assembly and functioning of the spine simulator. The in-silico development and validation of the 6 DOF load control system will allow seamless implementation of load control within the spine simulator. The ultimate outcome of this will be the ability to assess the behaviour of FSUs subjected to biofidelic loading conditions

Cells typically respond to a variety of geometrical cues in their environment, ranging from nanoscale surface topography to mesoscale surface curvature. The ability to control cellular organisation and fate by engineering the shape of the extracellular milieu offers exciting opportunities within tissue engineering. Despite great progress, however, many questions regarding geometry-driven tissue growth remain unanswered. Here, we combine mathematical surface design, high-resolution microfabrication, in vitro cell culture, and image-based characterization to study spatiotemporal cell patterning and bone tissue formation in geometrically complex environments. Using concepts from differential geometry, we rationally designed a library of complex mesostructured substrates (10. 1. -10. 3. µm). These substrates were accurately fabricated using a combination of two-photon polymerisation and replica moulding, followed by surface functionalisation. Subsequently, different cell types (preosteoblasts, fibroblasts, mesenchymal stromal cells) were cultured on the substrates for varying times and under varying osteogenic conditions. Using imaging-based methods, such as fluorescent confocal microscopy and second harmonic generation imaging, as well as quantitative image processing, we were able to study early-stage spatiotemporal cell patterning and late-stage extracellular matrix organisation. Our results demonstrate clear geometry-dependent cell patterning, with cells generally avoiding convex regions in favour of concave domains. Moreover, the formation of multicellular bridges and collective curvature-dependent cell orientation could be observed. At longer time points, we found clear and robust geometry-driven orientation of the collagenous extracellular matrix, which became apparent with second harmonic generation imaging after ∼2 weeks of culture. Our results highlight a key role for geometry as a cue to guide spatiotemporal cell and tissue organisation, which is relevant for scaffold design in tissue engineering applications. Our ongoing work aims at understanding the underlying principles of geometry-driven tissue growth, with a focus on the interactions between substrate geometry and mechanical forces

Introduction. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels. Method. Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques. Result. A 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial. Conclusion. A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration. The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

Introduction

Bereft of their optimal tissue context, cells lose their phenotype, function and therapeutic potential during in vitro culture. Despite the fact that in vivo cells are exposed simultaneously to multiple signals, traditional ex vivo cultures are monofactorial. With these in mind, herein we assessed the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human tenocyte, skin fibroblast and bone marrow mesenchymal stromal cell cultures.

Differences at motor control strategies to provide dynamic balance in various tasks in diabetic polyneuropatic (DPN) patients due to losing the lower extremity somatosensory information were reported in the literature. It has been stated that dynamics of center of mass (CoM) is controlled by center of pressure (CoP) during human upright standing and active daily movements. Indeed analyzing kinematic trajectories of joints unveil motor control strategies stabilizing CoM. Nevertheless, we hypothesized that imbalance disorders/CoM destabilization observed at DPN patients due to lack of tactile information about the base of support cannot be explained only by looking at joint kinematics, rather functional foot usage is proposed to be an important counterpart at controlling CoM. In this study, we included 14 DPN patients, who are diagnosed through clinical examination and electroneuromyography, and age matched 14 healthy subjects (HS) to identify control strategies in functional reach test (FRT). After measuring participants’ foot arch index (FAI) by a custom-made archmeter, they were tested by using a force plate, motion analysis system, surface electromyography and pressure pad, all working in synchronous during FRT. We analyzed data to determine effect of structural and functional foot pathologies due to neuropathy on patient performance and postural control estimating FAI, reach length (FR), FR to height (H) ratio (FR/H; normalized FR with respect to height), displacement of CoM and CoP in anteroposterior direction only, moment arm (MA, defined as the difference between CoP and CoM at the end of FRT), ankle, knee and hip joint angles computed at the sagittal plane for both extremities. Kinematic metrics included initial and final joint angles, defined with respect to start and end of reaching respectively. Further difference in the final and initial joint angles was defined as Δ. FAI was founded significantly lower in DPN patients (DPN: 0.3404; HS: 0.3643, p= <0.05). The patients’ FR, FR/H and absolute MA and displacement of CoM were significantly shorter than the control group (p= <0.05). Displacement of CoP between the two groups were not significant. Further we observed that CoM was lacking CoP in DPN patients (mean MA: +0.88 cm), while leading CoP in HS (mean MA: −1.59 cm) at the end of FRT. All initial angles were similar in two groups, however in DPN patients final right and left hip flexion angle (p=0.016 and p=0.028 respectively) and left ankle plantar flexion angle (p=0.04) were smaller than HS significantly. DPN patients had significantly less (p=0.029) hip flexion (mean at right hip angle, Δ=25.0°) compared to HS (Δ=33.53°) and ankle plantar flexion (DPN mean at right ankle angle, Δ=6.42°, HS mean Δ=9.07°; p=0.05). The results suggest that movement of both hip and ankle joints was limited simultaneously in DPN patients causing lack of CoM with respect to CoP at the end of reaching with significantly lower FAI. These results lead to the fact that cutaneous and joint somatosensory information from foot and ankle along with the structure of foot arch may play an important role in maintaining dynamic balance and performance of environmental context. In further studies, we expect to show that difference at control strategies in DPN patients due to restricted functional foot usage might be a good predictor of how neuropathy evolves to change biomechanical aspects of biped erect posture

Gradients of three-dimensional (3D) hierarchical tissues are common in nature and present specific architectures, as this is the case of the anisotropic subchondral bone interfaced with articular cartilage. While diverse fabrication techniques based on 3D printing, microfabrication, and microfluidics have been used to recreate tailored biomimetic tissues and their respective microenvironment, an alternative solution is still needed for improved biomimetic gradient tissues under dynamic conditions with control over pre-vasculature formation. Here, we engineered a gradient osteochondral human-based tissue with precise control over both cell/tissue phenotype and pre-vasculature formation, which opens-up possibilities for the study of complex tissues interfaces, with broader applications in drug testing and regenerative medicine. The fabrication of 3D gradients of microparticles was performed combining methacrylated gelatin (GelMA) and gellan gum (GG) (3:1, w:w ratio) with hydroxyapatite microparticles (HAp, 30% w/w). The mixing of the interface was controlled by the temperature of two polymeric layers, being the second added at 10 ºC higher than the first one. This subsequent addition of polymeric solutions at different temperatures promoted convection, which drove the microparticles through the interface from the first to the second layered gel forming the HAp gradient. After ionic and photo-crosslinking, the freezing step was programmed using an external cover of styrofoam forcing the ice crystals to grow linearly, generating an anisotropic architecture in a gradient scaffold. A dual-chamber microreactor device was designed (figure 1A) to culture fat pad adipose-derived stem cells and microvascular endothelial cells under two biochemical microenvironments. Using control over temperature and crosslinking, hydrogel-like structures were built in 3D anisotropic HAp gradients. Then, an in vitro osteochondral tissue model was obtained using a dual-chamber platform. Results showed a significant difference of SOX9 (p < 0.05), Osteocalcin and RUNX2 (p < 0.05) from the top to the bottom regions of the 3D gradient structures under dynamic conditions. Finally, a pre-vasculature was controlled over 7 days, stimulating the endothelization of the subchondral bone-like region 35% more (p < 0.05) when compared to the cartilage-like region. In this work, microparticle and biochemical gradients were fabricated into anisotropic architectures. The obtained outcomes enable the precise control of 3D gradients in programmable architectures, such as anisotropic structures, with broad applications in interfaced tissue engineering, regenerative medicine and drug testing

The development of functional biomaterials scaffolds for bone tissue engineering applications includes the control of specific biological and mechanical parameters that are involved in the growth of bone tissue in a way that mimics the physiological process of healing bone defects. Here, we report on the development of composite scaffolds made from biodegradable natural and synthetic biomaterials with characteristic architectural features, functionalized with the osteoinductive growth factor bone morphogenetic protein BMP-2, and evaluating their osteogenic response in static and dynamic cell culture systems. The results show that scaffold designing with advanced technologies combined with appropriate biochemical and mechanical stimulating factors, results to an enhanced proliferative and osteogenic/chondrogenic differentiation response of cells cultured on the developed scaffolds, and thus controlling the new tissue formation and reconstruction

Objectives. The objective of this study was to determine if combining variations in mixing technique of antibiotic-impregnated polymethylmethacrylate (PMMA) cement with low frequency ultrasound (LFUS) improves antibiotic elution during the initial high phase (Phase I) and subsequent low phase (Phase II) while not diminishing mechanical strength. Methods. Three batches of vancomycin-loaded PMMA were prepared with different mixing techniques: a standard technique; a delayed technique; and a control without antibiotic. Daily elution samples were analysed using flow injection analysis (FIA). Beginning in Phase II, samples from each mix group were selected randomly to undergo either five, 15, 45, or 0 minutes of LFUS treatment. Elution amounts between LFUS treatments were analysed. Following Phase II, compression testing was done to quantify strength. A-priorit-tests and univariate ANOVAs were used to compare elution and mechanical test results between the two mix groups and the control group. Results. The delayed technique showed a significant increase in elution on day one compared with the standard mix technique (p < 0.001). The transition point from Phase I to Phase II occurred on day ten. LFUS treatments significantly increased elution amounts for all groups above control. Delayed technique resulted in significantly higher elution amounts for the five-minute- (p = 0.004) and 45-minute- (p < 0.001) duration groups compared with standard technique. Additionally, the correlations between LFUS duration and total elution amount for both mix techniques were significant (p = 0.03). Both antibiotic-impregnated groups exhibited a significant decrease in offset yield stress compared with the control group (p < 0.001), however, their lower 95% confidence intervals were all above the 70 MPa limit defined by International Standards Organization (ISO) 5833-2 reference standard for acrylic bone cement. Conclusion. The combination of a delayed mix technique with LFUS treatments provides a reasonable means for increasing both short- and long-term antibiotic elution without affecting mechanical strength. Cite this article: Dr. T. McIff. Combination of modified mixing technique and low frequency ultrasound to control the elution profile of vancomycin-loaded acrylic bone cement. Bone Joint Res 2016;5:26–32. doi: 10.1302/2046-3758.52.2000412

Osteogenesis is key to fracture healing and osteointegration of implanted material. Modification of surfaces on a nanoscale has been shown to affect cell interaction with the material and can lead to preferential osteogenesis. We hypothesised that osteogenesis could be induced in a heterogeneous population of osteoprogenitor cells by circular nanopits on a material surface. Furthermore, we intended to assess any correlation between nanopit depth and osteoinductive potential. The desired topographies were embossed onto polycaprolactone (PCL) discs using pre-fabricated nickel shims. All pits had a diameter of 30μm and investigated pit depths were 80nm, 220nm and 333nm. Scanning electron microscopy confirmed successful embossing and planar controls were shown to be flat. A bone marrow aspirate was obtained from the femoral neck of a healthy adult undergoing a hip replacement. After establishing a culture, cells were seeded onto the PCL discs, suspended in basal media and incubated. Samples were fixed and stained after three and 28 days. Cells were stained for the adhesion molecule vinculin after three days. Lowest concentrations of vinculin were seen in the planar control group. Osteoprogenitor cells on the shallowest pits, 80nm, had larger and brighter adhesion complexes. After 28 days, osteocalcin and osteopontin expression were used as markers of cell differentiation into an osteoblastic phenotype. 220nm deep pits consistently produced cells with the highest concentrations of osteopontin (p = 0.017) with a similar trend of osteocalcin expression. Cells on all topographies had higher expression levels than the planar controls. We demonstrated stimulation of osteogenesis in a heterogeneous population of osteoprogenitor cells. This cell mix is similar to that present in fracture healing and after reaming for intramedullary devices or uncemented implants. All nanopit depths gave promising results with an optimum depth of 220nm after 28 days

Background. Hip arthroplasties are associated with high postoperative pain scores. In some reports, moderate to severe pain was 58% on the first day postoperatively in total hip replacements (THRs). Several techniques are currently used at our institution to tackle acute pain following THRs. These include: 1) Spinal anaesthetic (SA) with Diamorphine only; 2) General anaesthetic (GA) only; 3) SA with local infiltration anaesthetic mixture 1 (LIA1,). Mixture 1 consisted of ropivacaine, adrenaline, and ketorolac; 4) SA with LIA mixture 2 (LIA2). Mixture 2 consisted of bupivacaine and adrenaline; 5) SA with LIA1 and PainKwell pump system. In this study we report on the techniques of acute pain control following THR at our regional centre for elective primary THRs. Methods. Between June 2011 and July 2014, 173 consecutive patients undergoing primary THR using the posterior approach were prospectively followed up. Group 1. GA only. 31 patients, Group 2. SA only. 37 patients, Group 3. SA plus LIA1 only. 38 patients, Group 4. SA plus LIA2 only, 34 patients, Group 5. SA plus LIA1 plus PainKwell Pump System for 48 hours. 33 patients. Results. Fewer patients required opiate analgesia when LIA plus PainKwell pump system was used compared to the other groups. The highest significance was at 0–12 hrs for patients requiring up to 20mg morphine usage (χ2(2) = 46.713, p = 0.000); and 0–12hrs for patients requiring 30mg morphine usage (χ2(2) = 46.310, p = 0.000). There were no infections, DVTs or PEs in any group. One patient in group 3 suffered a stroke (ASA 4). A Kruskal-Wallis H test also showed that there was a statistically significant difference in morphine usage across groups 1, 2, 3, 4, and 5. Conclusion. We recommend the use of LIA with PainKwell pump system continuous infusion as an efficacious method to control pain following THR

We have developed precision-engineered strontium eluting nanopatterned surfaces. Nanotopography has been shown to increase osteoblast differentiation, and strontium is an element similar to calcium, which has been proven to increase new bone formation and mineralization. This combination has great potential merit in fusion surgery and arthroplasty, as well as potential to reduce osteoporosis. However, osteoclast mediated osteolysis is responsible for the aseptic failure of implanted biomaterials, and there is a paucity of literature regarding osteoclast response to nanoscale surfaces. Furthermore, imbalance in osteoclast/osteoblast resorption is responsible for osteoporosis, a major healthcare burden. We aimed to assess the affect of strontium elution nanopatterned surfaces on osteoblast and osteoclast differentiation. We developed a novel human osteoblast/osteoclast co-culture system without extraneous supplementation to closely represent the in vivo environment. We assessed the surfaces using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using polymerase chain reaction (PCR). In complex co-culture significantly increased osteoblast differentiation and bone formation was noted on the strontium eluting, nanopatterned and nanopatterned strontium eluting surfaces, suggesting improved osteointegration. There was a reduction in macrophage attachment on these surfaces as well, suggesting specific anti-osteoclastogenic properties of this surface. Our results show that osteoblast and osteoclast differentiation can be controlled through use of nanopatterned and strontium eluting surface features, with significant bone formation seen on these uniquely designed surfaces

Aim. To control the growth and function of osteoblasts on Titanium alloy surfaces produced by electrochemical patterning. Methods. Samples of Ti6Al4V were prepared with three different finishes; no surface preparation following machining, polishing on a grinding wheel with sequential grit papers up to 4000 to achieve a mirror finish and treatment in a flat electrochemical cell with a 3M sulphuric acid in methanol using 9V supplied over 60 seconds to produce a surface with defined nano/microscale roughness. Glass coverslips were used as control surfaces. Surfaces were seeded with primary rat calvarial osteoblasts and incubated in Dulbecco's Modified Eagle Medium with 10% (v/v) sera for 24 hours before fixing and performing immunofluorescence staining with anti-vinculin antibody. Photomicrographs of the surfaces were analysed with Image J and analySIS FIVE programs. Results for cell number, cell area, focal adhesion area and polarity (lack of roundness) were analysed (using the Mann Whitney test) for ANOVA using SPSS. Results. Cells adhered to all surfaces with the most cells on the polished surface and the fewest on the glass and 9V60s surfaces. There were significant differences in cell number only between the polished surface and the glass control (p=0.026) and the 9V60s surface (p=0.006). Cells grown on the glass control surfaces exhibited the largest areas (mean = 840micron2) whilst those on the machined surface were the smallest (mean = 601micron2). A significant difference in cell area was seen between the machined and polished surfaces (p=0.025). The area of the focal adhesions was significantly different between the cells on 9V60s surface and the glass control (p=0.004), machined (p=0.003) and polished surfaces (p=0.006). Significant differences in polarity were seen between the cells on machined surface and the glass control (p=0.004), polished (p=0.004) and 9V60s surfaces (p=0.004). Discussion. Differences in cell numbers on glass and two of the Ti surfaces may be explained by the smooth nature of the glass coverslips in comparison to the nanoscale topography on the polished and 9V60s treated surfaces. Cell area was noted to be different between the machined and smoother polished surface. This may be explained by the grooves present on the machined surfaces preventing normal cell spreading by the process of contact guidance. There was a marked difference in polarity between the most polarised cells on the machined surface and the more rounded cells on the smoother surfaces, again consistent with the behaviour of contact guidance, with cells growing in the direction of the surface grooves. Focal adhesions present on the 9V60s treated surface were very small in comparison to those on other surfaces. Several features of implant surfaces may affect osteoblast growth, including surface roughness, chemical composition, surface charge and surface energy. These features influence the adsorption of proteins onto the surfaces, in turn influencing the growth and behaviour of the adherent cell population. Conclusion. Mechanical and electrochemical treatment of titanium alloy can significantly affect the growth and behaviour of osteoblasts grown on the surface. This has potential applications in arthroplasty and fracture fixation

Summary. This study helps to elucidate how ColVI and Dcn within the pericellular matrix (PCM) of differentiating hMSCs directly impacts dynamic cytoskeletal response to load, and demonstrates an important role for the PCM in mechanotransduction during chondrogenesis. Introduction. Mechanosignaling events in differentiating human mesenchymal stem cells (hMSCs) are dependent on their temporally changing micromechanical environment and their dynamic cytoskeleton. During chondrogenic differentiation, hMSCs develop a matrix composed of type VI collagen (ColVI) and proteoglycans such as decorin (Dcn). We have previously demonstrated that this developing PCM is important in cellular mechanotransduction. The aim of this study was to determine the functional roles of ColVI and Dcn in modulating load-induced changes in the organization of vimentin intermediate filaments (VIF), actin microfilaments (AM), and vinculin. Methods. hMSCs were transduced with shRNA targeting either col6a1 (shColVI) or dcn (shDcn) and then cultured in 2% alginate beads for 14 days in chondrogenic media. GFP-transduced hMSCs were cultured in parallel. Cells with their intact PCM were isolated with 100mM sodium citrate, 30mM EDTA, and re-embedded in alginate discs. These hMSC-alginate constructs underwent unconfined compression at 0.1Hz for 1 hour from 0–10% strain. Free Swelling (FS) and loaded discs were fixed either immediately following (0hr) or 4 hours post-load. Discs were cryosectioned and fluorescently labeled for VIF, AM, or vinculin with DAPI counterstain. Confocal image-stacks were collected and corrected total cell fluorescence (CTCF) per area was calculated using ImageJ (NIH) to quantify cytoskeletal organization. Results. VIF fluorescence showed a dynamic cytoskeletal response to load in non-infected and GFP-transduced controls with an increase in intensity. In both ColVI and Dcn knockdown groups, VIF exhibited higher fluorescence in FS, which then didn't significantly change following load, possibly due to its higher baseline concentration. AM showed a similar response to load in shColVI knockdown samples, with a significantly higher intensity in FS samples, which wasn't affected by loading and remained cortically localised in all samples. In shDcn samples, there was a significant increase in actin content immediately following load, similar to control. Vinculin staining showed significantly lower staining intensity in shColVI knockdown samples than non-infected and GFP-transduced samples at all timepoints, and exhibited a different trend in response to loading. Following loading, vinculin was diffusely stained across the cytoskeleton in all samples. Discussion. This study demonstrates through targeted shRNA knockdown that the involvement of ColVI and Dcn in mechanosignaling events of differentiating hMSCs could be due to their interactions with and control over the dynamic cytoskeleton. Vimentin and actin have previously been shown to contribute to the viscoelastic mechanical properties of hMSCs and are important in the ability for cells to resist load. Our experiments indicate that specific components in the PCM can affect cytoskeletal dynamics, with knockdown samples lacking the significantly dynamic response to load seen in control samples. Actin cytoskeletal intensity and vinculin intensity show an inverse relationship to load. Since vinculin mediates interactions between actin cytoskeleton and integrins at focal adhesions, these data suggest that ColVI and Dcn regulation of cytoskeletal response may be governed more by the changing external micromechanical environment, rather than altered cell-matrix interactions. Further studies are needed to elucidate the roles of ColVI and Dcn in downstream intracellular mechanosignaling events of differentiating hMSCs

There is a growing interest in the development of tissue engineering (TE) therapies to repair damaged bone. Among the scaffolds for TE applications, injectable hydrogels have demonstrated great potential as three-dimensional cell cultures in bone TE, owing to their high water content, porous structure that allows cell transplantation and proliferation, similarity to the natural extracellular matrix and ability to match irregular defects. We investigated whether fibrin-based hydrogels capable of transducing near infrared (NIR) energy into heat can be employed to lead bone repair. Hollow gold nanoparticles with a plasmon surface band absorption at ∼750 nm, a NIR wavelength within the so called “tissue optical window”, were used as fillers in injectable fibrin-based hydrogels. These composites were loaded with genetically-modified cells harbouring a heat-activated and rapamycin-dependent gene circuit to regulate transgenic expression of the reporter gene firefly luciferase (fLuc). NIR-responsive cell constructs were injected to fill a 4 mm diameter critical-sized defect (CSD) in the parietal bone of mouse calvaria. NIR-irradiation in the presence of rapamycin triggered a pattern of fLuc activity that faithfully matched the illuminated area of the implanted hydrogel. Having shown that this platform can control the expression of a transgene product, we tested its effectiveness on regulating the secretion of transgenic bone morphogenetic protein 2 (BMP-2) from NIR-responsive hydrogels implanted in CSD. The spatiotemporal pattern of transgenic BMP-2 secretion induced by NIR-irradiation in the presence of rapamycin significantly stimulated bone regeneration from the edge of osteotomy in the CSD practiced, validating the therapeutic approach

Introduction. Bone-anchored devices have been used as skin-crossing conduits to record neuromuscular signals in sedated animals. Long-term recordings from cognisant subjects must be assessed. Hypothesis A bone-anchored device is suitable as a conduit for epimysial EMG (Electromyogram) recordings and is reliable in the long-term. Methods. The bone-anchored device was implanted into the medial aspect of an ovine tibia (n=1), and the epimysial electrode was sutured onto the peroneus tertius muscle. Epimysial and Surface EMG signals were recorded for 12 weeks. Results. The signal-to-noise ratio (SNR) was greater for epimysial (5.1) than surface electrodes (1.6). SNR deteriorated near the end of 12 weeks, due to debris in an external connector. Discussion and Conclusion. Implanted electrodes improve SNR, selectivity, signal reliability and reduce cross-talk. Bone-anchored devices allow hard-wired connections without infection or fatigue at the skin-interface. Hard-wired connections will enable more advanced prosthetic control. This is the first known use of a bone-anchored device to acquire physiological signals from a cognisant subject

Introduction. Changes in central nervous system (CNS) pathways controlling trunk and leg muscles in patients with low back pain(LBP) and lumbar radiculopathy have been observed and this study investigated whether surgery impacts upon these changes in the long term. Methods. 80 participants were recruited into the following groups: 25 surgery(S), 20 chronic LBP(CH), 14 spinal injection(SI), and 21 controls(C). Parameters of corticospinal control were examined before, at 6, 26 and 52 weeks following lumbar decompression surgery and equivalent intervals. Electromyographic(EMG) activity was recorded from tibialis anterior(TA), soleus(SOL), rectus abdominis(RA), external oblique(EO) and erector spinae(ES) muscles at the T12&L4 levels in response to transcranial magnetic stimulation of the motor cortex. Motor evoked potentials (MEP) and cortical silent periods(cSP) recruitment curves(RC) were analysed. Results. Trunk muscles in all patients had reduced raw EMG (P<0.001), increased motor thresholds (MTh;P<0.001) and MEP RC slopes. MTh in ESL4 correlated with back pain in all patients (r=0.201, P=0.016) and soleus MTh laterality with disability in surgery patients (r=0.49, P=0.018). S&SI patients displayed bilaterally increased soleus cSP (p<0.001), MEP latencies on the painful side (P<0.001), and cSP asymmetry (cSPA;P<0.001). cSPA resulted from abnormal soleus late responses on the painful side, indicating compromised agonist-antagonist control in patients with radiculopathy. In contrast to SI, surgery significantly reduced soleus cSPA and MEP latencies at 6 weeks (P≤0.034). Discussion. These results show long term changes in CNS control of trunk and leg muscles in radiculopathy and LBP, which are only partly reversed by surgery, and may provide future therapeutic targets to address the altered inhibitory processes within the brain. No conflicts of Interest. Sources of funding: The DISCS foundation. This abstract has not been previously published in whole or substantial part nor has it been presented previously at a national meeting

Axial musculoskeletal control (AMC) is a widely used concept and has been shown to be an important factor in physical performance, the pathophysiology of back pain and other MSK conditions. However, there is no agreement on a definition of AMC, nor a validated test for AMC and its application in clinical practice. Our aim was to develop a test for AMC using the Delphi method from a panel of experts with video and analysis of the footage. We found that the most commonly used tests were the maintenance of neutral pelvic position in single leg stance, single leg stance with eyes closed and single leg squat. We aim to further validate our findings by comparing this to surface EMG recordings and centre of gravity measurements in stress situations

Current cell-based tissue engineering strategies have limited clinical applicability due to the need for large cell numbers and prolonged culture periods that lead to phenotypic drift. In vitro microenvironmental modulators have been proposed to mimic the native tendon. Standard in vitro culture conditions result in delayed extracellular matrix (ECM) deposition, impairing the development of scaffold-free approaches. ECM deposition can be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the milieu of multicellular organisms. We assessed a multifactorial biophysical approach, using MMC and mechanical loading, on different cell sources to determine their suitability for in vitro fabrication of tendon-like tissue. Human dermal fibroblasts (DFs), tenocytes (TCs) and bone marrow mesenchymal stem cells (BMSCs) were cultured with MMC under static and uniaxial strain culture conditions. TCs and DFs exhibited alignment perpendicular to the load, whilst BMSCs did not show preferential alignment. When MMC was used, DFs and BMSCs showed increased deposition of collagen I, the main component in tendon ECM. DFs presented ECM composition similar to TCs with collagen types III, V and VI present. Gene expression analysis revealed upregulation of tenogenic markers by TCs and DFs, such as scleraxis and thrombospondin-4, under both loading and MMC. The combined use of MMC and mechanical stimulation is suitable for TCs phenotype maintenance and can modulate the phenotype of DFs and BMSCs differentially. This study provides insight into response of different cell sources to biophysical cues and contributes to further development of cell therapies for tendon repair and regeneration.

During in vitro sub-culturing, tenocytes lose their phenotype which ultimately affects their functioning. As spindle-shaped fibroblasts, tenocytes have a unique thin elongated phenotype and they possess more spread-out shape through phenomena named dedifferentiation1. Given the link between cell shape and cell function, in this study, we first aimed to dedifferentiate tenocytes through in vitro sub-culturing in order to have a model system for dedifferentiation. For this, we isolated human flexor tendon cells from healthy female flexor digitorum longus and seeded at 5000 cells/cm² cell density, passaged every two days for six passages. In order to assess cell phenotype, we fixed with 4% paraformaldehyde and stained with phalloidin and DAPI to visualize the actin cytoskeleton and DNA respectively. We noted that in each passage, cells lost their spindle-shaped phenotype and became more pancake-shaped. At passage 1 and 2, the main cell phenotype is spindle-shaped. However, as the cells are further passaged, the phenotype of the cell population becomes more heterogeneous and at passage 5 and 6, they already display a more spread-out shape. Based on these results, we further hypothesized that they can be re-differentiated through matrix-mediated mechano-transduction and regain their morphology and function. For this aim, we generated decellularized tendon from porcine Achilles tendon and setup a mechanical loading system where we can provide mechanical loadings at physiological levels. This system will provide a new approach on in vitro tenocyte culturing.

Summary. Randomised controlled study evaluating new bone formation in vivo in fracture non-unions by bone marrow derived stromal cells (BMSC). These cells do not show statistically significant new bone formation. Age of the patient during fracture, diabetes and doubling time had been observed to be correlated with fracture healing. Introduction. Regenerating new bone by cell therapy could provide therapeutic options in many conditions such as fracture non-unions and osteo-chondral defect regeneration in advance OA. In this randomised controlled study we evaluated the efficacy of new bone formation by bone marrow derived stromal cells (BMSC) in patients with non-union. Methods. An ethically approved and adequately powered single centre randomised control trial recruited 35 patients for treatment of non-unions with BMSC. Bone marrow was harvested and autologous BMSC were culture expanded in autologous serum at our local MHRA-licensed facility (Oscell, Oswestry, UK). Following selection by adherence and in vitro culture expansion using autologous serum, cells in serum and serum alone was randomised for insertion at one of the two fracture sides by StratOs® computer software. Patients and the operating surgeon were blinded to the side of cell insertion. Such method of randomisation created internal controls at the fracture sites- one side receiving the cell (‘test side’) and other, not (‘control’). Serial radiographs extending up to an average of twelve months were evaluated by four independent assessors blinded to side of cell insertion. Callus formation and bridging of fracture was compared for ‘test’ and ‘control’ side. Radiological and clinical outcome at final follow-up was also noted. Results. Thirty five patients were recruited (21 males, 14 females; mean age 51.2±13.2SD). The mean duration of non-union was 3±2SD years, with a mean 3.5 (range 1–12) surgical interventions prior to BMSC insertion. Five patients had diabetes. New callus formation and fracture bridging was slow, with no significant difference between the cell-insertion and control side although a substantial improvement in fracture bridging/formation of new callus was noted at 9–12 months. Fracture union was achieved in 21 patients at final follow-up with failure to progress to union in 14 patients. Age at accident, having diabetes and cell doubling time during culture predicted union (r2=0.63, p=0.017). There was no reported adverse effects from the trial. Conclusion. The study concluded that patient biology predicts the final outcome in cases with non-union of fracture. Slower doubling time during in vitro expansion can be significantly correlated with failure to unite in addition to diabetes and age of the patient. BMSC's are safe option for cell therapy in a setting of non-union although it failed to show statistically significant difference of new bone formation or fracture bridging for up to one year