Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development

Abstract. BACKGROUND. Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. OBJECTIVE. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. RESULTS. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. CONCLUSION. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. Objectives. Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype. Results. Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNAseq revealed that there were 2,085 highly significant upregulated genes in 3D matrices compared to TCP. Notably, >90% of highly expressed genes (including FOSB, FOS and TNFAIP6) were shared in all hydrogels. Gene ontology confirmed the TNF signalling pathway among the most significantly represented. Protein-protein interaction predictions identified TNF-alpha/NF-kappa B and AP1 pathways as differentially influenced by the hydrogel environment. Using inhibitors to these pathways, NFkB, but not AP1, impacted on the upregulation of TNFAIP6 and CXCL10 in 3D culture. Conditioned media from these studies was added to cultures of human monocytes with distinct changes in the resulting macrophage phenotype. MSCs in a 3D environment promoted a greater acquisition of the M2 repair macrophage phenotype and impacted on the numbers of pro-inflammatory M1 macrophages. Conclusion. These data provide further evidence that the immunomodulatory action of human MSCs can be influenced by the surrounding structural environment. These observations have significance for understanding the events that following skeletal injury and the potential to be exploited in preconditioning MSCs for cell therapy

The management of bone defects and impaired fracture healing remains one of the most challenging clinical problems. Several treatments exist to aid in the healing of large bone defects, including biologics such as recombinant human bone morphogenetic protein-2 (BMP-2), yet all have met with limited success. Regeneration of bone requires a coordinated network of molecular signals where the local mechanical environment plays a major role in the success of the healing process. The mechanical environment itself is determined by the stiffness of the implant used to stabilize the fracture and weight-bearing, and if fixation is either too flexible or too rigid the healing might fail. The hypothesis is that the healing of large-segmental bone defects and fractures can be accelerated by the imposition of an appropriate mechanical environment. An overview of the progress made in this research area on how the amount of rhBMP-2 could be reduced and its effectiveness increased by providing an optimized mechanical environment to achieve bone union will be presented. Additionally, the latest findings of improved fracture healing through the manipulation of fixation stability introducing a potential clinical strategy to improve the healing outcome of unstable fractures, particularly for non-unions through increased stabilization, will be discussed

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons. The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment

Introduction. Civilian fractures have been extensively studied with in an attempt to develop classification systems, which guide optimal fracture management, predict outcome or facilitate communication. More recently, biomechanical analyses have been applied in order to suggest mechanism of injury after the traumatic insult, and predict injuries as a result of a mechanism of injury, with particular application to the field so forensics. However, little work has been carried out on military fractures, and the application of civilian fracture classification systems are fraught with error. Explosive injuries have been sub-divided into primary, secondary and tertiary effects. The aim of this study was to 1. determine which effects of the explosion are responsible for combat casualty extremity bone injury in 2 distinct environments; a) in the open and b) enclosed space (either in vehicle or in cover) 2. determine whether patterns of combat casualty bone injury differed between environments Invariably, this has implications for injury classification and the development of appropriate mitigation strategies. Method. All ED records, case notes, and radiographs of patients admitted to the British military hospital in Afghanistan were reviewed over a 6 month period Apr 08-Sept 08 to identify any fracture caused by an explosive mechanism. Paediatric cases were excluded from the analysis. All radiographs were independently reviewed by a Radiologist, a team of Military Orthopaedic Surgeons and a team of academic Biomechanists, in order to determine the fracture classification and predict the mechanism of injury. Early in the study it became clear that due to the complexity of some of the injuries it was inappropriate to consider bones separately and the term ‘Zone of Insult’ (ZoI) was developed to identify separate areas of injury. Results. 62 combat casualties with 115 ZoIs (mean 1.82 zones) were identified in this study. 34 casualties in the open sustained 56 ZoIs (mean 1.65); 28 casualties in the enclosed group sustained 59 ZoIs (mean 2.10). There was no statistical difference in the mean ZoIs per casualty in the open vs enclosed group (Student t-test, p=0.24). Open fractures were more prevalent in the open group compared to the enclosed group (48/59 vs 20/49, Chi-squared test p<0.001). Of the casualties in the open, 1 zone of injury was due to the primary effects of blast, 10 a combination of primary and secondary blast zones, 23 due to secondary effects and 24 from the tertiary effects of blast. In contrast, there were no primary or combined primary and secondary blast zones and only 2 secondary blast zones in the enclosed group. Tertiary blast effects predominated in the enclosed group, accounting for 96% of injury zones (57/59). Analysis of the pattern of injury revealed that there were a higher proportion of lower limb injuries in the Enclosed group (54/59) compared to the Open group (40/58, Chi-squared p<0.05). In the Open group the mechanism of lower limb injury was more evenly distributed amongst mixed primary and secondary blast effects (10), secondary (10) and tertiary (20). In the enclosed group, lower limb injuries were almost exclusively caused by tertiary blast effects (47/48). A similar pattern was also seen in the Upper limb with 4/5 in the enclosed group was injured by tertiary effects compared to 4/18 in the Open Group. In the open group fragmentation injury was the predominant cause of injury (13/18). Conclusions. This data clearly demonstrates two distinct injury groups based upon the casualties' environment. The enclosed environment afforded by buildings and vehicles appears to mitigate the primary and secondary effects of the explosion. However, tertiary blast effects were the predominant mechanism of injury, with severe axial loading to the lower extremity being a characteristic of the fractures seen. In contrast, secondary fragments from the explosion were more likely to result in fractures of casualties caught in the open. The development of future mitigation strategies must be focused on reducing all the different mechanisms of injury caused by an explosion. This will require a better understanding on the effects of bone in high strain environments. This method of forensic biomechanics involving clinicians and engineers, combined with accurate physical and numerical simulations can form the basis in reducing the injury burden to the combat soldier

Introduction. One method of surgical site infection prevention is lowering intraoperative environmental contamination. We sought to evaluate our hospitals operating room (OR) contamination rates and compare it to the remainder of the hospital. We tested environmental contamination in preoperative, intraoperative and postoperative settings of a total joint arthroplasty patient. Materials & Methods. 190 air settle plates composed of trypsin soy agar (TSA) were placed in 19 settings within our hospital. Locations included the OR with light and heavy traffic, with and without masks, jackets, and shoe covers, sub-sterile rooms, OR hallways, sterile equipment processing center, preoperative areas, post-anesthesia care units, orthopaedic floors, emergency department, OR locker rooms and restrooms, a standard house in the local community, and controls. The plates were incubated in 36 degrees celsius for 48 hours and colony counts were recorded. Numbers were averaged over each individual area. Results. The highest CFU was the OR locker room at 28 CFU/plate/hr. Preoperative & post anesthesia care unit holding areas were 7.4 CFU & 9.6 CFU, respectively. The main orthopaedic surgical ward had 10.0 CFU/plate/hr, while the VIP hospital ward had 17.0 CFU/plate/hr. The OR environment all had low CFUs. A live OR had slightly higher CFUs than ones without OR personnel. The OR sub-sterile room had 5.2 CFU/plate/hr, and the OR hallway had 11.2 CFU/plate/hr. The local community household measured 5.6CFU/plate/hr. Discussion. In comparison to the local community household, the OR locker room, restrooms, hospital orthopaedic wards, ED, pre-operative holding, PACU and OR hallway all had higher airborne contamination than the local household in our surrounding community. We were surprised to find some areas with high rates of contamination. Our hospital has since increased environmental cleaning and monitoring of these areas with improved effect. Based on our results, we can recommend environmental sampling as a simple, fast, inexpensive tool to monitor airborne contamination

Unresolved inflammatory processes in tendon healing have been related to the progression of tendinopathies. Thus, the management of tendon injuries may rely on cell-based strategies to identify and modulate tendon inflammatory cues. Pulsed electromagnetic field (PEMF) has been approved by FDA for orthopedics therapies and has been related to a reduction in pain and to improve healing. However, the influence of PEMF in tendon healing remains largely unknown. Human tendon resident cells (hTDCs) were cultured in an inflammatory environment induced by exogenous supplementation of IL-1β and their response assessed after exposure to different PEMF treatments. This study demonstrates that IL-1β induced up-regulation of pro-inflammatory factors (IL-6 and TNFα) and extracellular matrix components (MMP−1, −2, −3) whereas reduces the expression of TIMP-1, suggesting IL-1β as a candidate inflammation model to study hTDCs response to inflammation cues. Moreover, in both homeostatic and inflammatory environments, hTDCs respond differently to PEMF treatment suggesting that cells are sensitive to magnetic field parameters such as strength (1.5 – 5mT), frequency (5–17Hz) and duration (10–50% duty cycle, dc). Among the conditions studied, PEMF treatment with 4mT/5Hz/50%dc suppresses the inflammatory response of hTDCs to the IL-1β stimulation, as evidenced by the decreases amount of IL-6, TNFα and downregulation of MMP-1, −2, −3 and COX-2, IL-8, IL-6, TNFα genes. These results demonstrate the potential of PEMF, in particular 4mT/5Hz/50%dc PEMF in treating tendon inflammation suppressing the inflammatory stimulation induced by IL-1β, which may be beneficial for tendon healing strategies

Introduction

Most western countries have implemented fast-track hip fracture aiming at surgery within 24 hours, since the mortality rate hereafter rises markedly.

In Greenland, it is not achievable to operate within 24 hours. Arctic people live in sparsely populated areas and Greenland's population is scattered along the vast coastline. All patients must be chartered to Nuuk by airplane which can take up till several days to weeks, due to logistics and the Arctic weather. This presents a challenge regarding adhering to western guidelines. The operative delay may be acceptable though, as it is the impression that the Greenlandic population survives and endures better than patients of western populations.

However, as data are lacking, we aimed to describe mortality among hip fracture patients in Greenland taking frailty and comorbidities into account.

Several previous studies have examined the mechanical environment in the femur using computational modelling. In particular the proximal femur has been extensively studied using finite element (FE) analyses. This study considers the issues associated with modelling with special interest in the distal femur. FE models require appropriate input on the geometry of the system being considered, material properties of different components, loading regimes and boundary conditions (i.e. the manner in which the system is supported). This study focuses on the last two of the above. A number of models with variable levels of complexity; and different boundary and loading conditions were considered. The simplest loading and boundary conditions considered comprised load applications at the tibio-femoral joint with the proximal femur artificially restrained. More complex models had the femur fully supported on muscles and ligaments. In each case the stress-strain environment in the femur was examined. The results show that the sophistication of the model needs to be based on the answers being sought from the analysis. Some good predictions on the mechanical environment can be made with relatively crude models. For example the stress-strain behaviour in the vicinity of the knee joint was found to be reasonably well predicted by the model that was artificially restrained in the mid-femoral region. Further while different models can be used for comparing different scenarios (e.g. forces during the gait cycle) true quantitative measures are strongly dependent on experimental loading data. The study also shows that it is important to generate and evaluate models of increasing complexity in order to maintain transparency with respect to the influence of different parameters associated with loading and boundary conditions

Background. Intervertebral disc cells exist in a challenging physiological environment. Disc degeneration occurs early in life implying that disc cells may no longer be able to maintain a functional tissue. We hypothesise that disc cells have a stress response different from most other cells because of the disc environment. We have compared the stress response of freshly isolated and cultured bovine nucleus pulposus (NP) cells with bovine dermal fibroblasts, representative of cells from a vascularised tissue. Methods. Freshly isolated and passaged bovine NP cells and dermal fibroblasts were cultured for 3 days then subjected to either thermal stress at 45°C for 1h followed by recovery times of 6, 24 and 48h or nutrient stress involving culture without serum for 6, 24 and 48 h. At each time point, cell number and viability were assessed and heat shock protein 70 (Hsp70) measured in cell lysates by an enzyme-linked immunosorbent assay. Results. In response to thermal stress, both freshly isolated and passaged dermal fibroblasts and also passaged NP cells showed a rapid elevation of Hsp70. In contrast, freshly isolated NP cells exhibited an attenuated Hsp70 response. With nutrient stress, Hsp70 increased with time in all dermal fibroblasts and passaged NP cells after 24 h, but freshly isolated NP cells responded differently again, producing less Hsp70 than controls. Conclusion. Freshly isolated bovine NP cells have a reduced response to applied stresses. This pilot study suggests that NP disc cells may have adapted to their physiologically challenging in vivo environment by attenuating their response to environmental stress. No conflicts of interest. Sources of Funding: The Wolfson Charitable Trust and Genodisc (EC's 7. th. Framework Programme (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626). This abstract has not been previously published in whole or substantial part nor has it been presented previously at a national meeting

Study Aim. Femoral components used in total knee arthroplasty (TKA) are primarily designed on the basis of kinematics and ease of fixation. This study considers the stress-strain environment in the distal femur due to different implant internal geometry variations (based on current industry standards) using finite element (FE) analyses. Both two and three dimensional models are considered for a range of physiological loading scenarios – from full extension to deep flexion. Issues associated with micro-motion at the bone-implant interface are also considered. Materials and methods. Two (plane strain) and three dimensional finite element analyses were conducted to examine implant micro-motions and stability. The simple 2D models were used to examine the influence of anterior-posterior (AP) flange angle on implant stability. AP slopes of 3°, 7° and 11° were considered with contact between bone and implant interfaces being modeled using the standard coulomb friction model. The direction and region of loading was based on loading experienced at full extension, 90° flexion and 135° flexion. Three main model variations were created for the 3D analyses, the first model represented an intact distal femur, the second a primary implanted distal femur and the third a distal femur implanted with a posterior stabilising implant. Further each of the above 3D model sets were divided into two group, the first used a frictional interface between the bone and implant to characterise the behavior of uncemented implants post TKA and the second group assumed 100% osseointegration had already taken place and focused on examining the subsequent stress/strain environment in the femur with respect to different femoral component geometries relative the intact distal femur model. Results and Discussion. Analyses indicate a trend relating the slope of the anterior-posterior (AP) flange to implant loosening at high flexion angles for uncemented components. Once cemented, this becomes less important. Results from the 3D analyses show that the posterior stabilising implant causes stress concentrations which can lead to bicondylar fatigue fracture. All femoral components cause stress shielding in cancellous bone particularly when they are fully bonded. Investigations into implant micromotion show that revision implants with box sections provided more resistance to micromotion than the pegged primary implants. However for the gait cycle tested the maximum recorded micromotion of both implants was well within acceptable levels for osseointegration to occur

Introduction and Objective

It is widely accepted that interfragmentary strain stimulus promotes callus formation during secondary bone healing. However, the impact of the temporal variation of mechanical stimulation on fracture healing is still not well understood. Moreover, the minimum strain value that initiates callus formation is unknown. The goal of this study was to develop an active fixation system that allows for in vivo testing of varying temporal distribution of mechanical stimulation and that enables detection of the strain limit that initiates callus formation.

Mesenchymal stem cells (MSCs) are tissue-resident stroma cells capable of modulating immune cells through the secretion of paracrine factors. However, the comparison of MSCs potential, from different sources and submitted to hypoxia within a 3D scaffold, in secreting pro-healing factors has never been investigated. With a chemical composition similar to type I collagen, a major component of connective tissues retrieved in dental pulp, bone and umbilical cord, Hemocollagene® haemostatic foam presented porous and interconnected structure (> 90%) and a relative low elastic modulus of around 60 kPa. All these criteria meet basic requirements for tissue engineering based material. Herein, we assessed and compared the effect of hypoxia (3% O₂) on the regulation and release of pro-angiogenic factors (VEGF, b-FGF and IL-8) from bone marrow (BM), Wharton's jelly (WJ) and dental pulp (DP) derived MSCs cultured in Hemocollagene®. After 10 days of culture, qRT-PCR analysis showed an up-regulation of b-FGF and VEGF mRNA in BM- and WJ-derived MSCs, but not in DP-derived MSCs. Furthermore, hypoxia highly up-regulated IL-8 expression in WJ-derived MSCs and moderately in both BM and DP-derived MSCs. In contrast, ELISA analysis showed a higher amount of VEGF and IL-8 in supernatant provided from DP-derived MSCs culture compared to BM and WJ-derived MSCs. B-FGF was not detected whatever the experimental condition. In conclusion, MSCs derived from several tissues were able to release pro-angiogenic factors under hypoxic conditions. There was no clearly superior type of MSCs for therapeutic use, however DP-derived MSCs are likely to be more advantageous.

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion.

Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue.

It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro.

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

Poor tendon repair is an unsolved issue in clinical practice, due to complex tendon structure. Tendon stem/progenitor cells (TSPCs) play key roles in homeostasis, regeneration, and inflammation regulation in acute tendon injuries, and rely on TGF-β signaling for recruitment into degenerative tendons. In this study, we aimed to develop an in vitro model for tenogenesis adopting a dynamic culture of a fibrin 3D scaffold, bioengineered with human TSPCs collected from both healthy and tendinopathic surgery explants (Review Board prot./SCCE n.151, 29 October 2020). 3D culture was maintained for 21 days under perfusion provided by a custom-made bioreactor, in a medium supplemented with hTGF-β1 at 20 ng/mL. The data collected suggested that the 3D in vitro model well supported survival of both pathological and healthy cells, and that hTGF-β signaling, coupled to a dynamic environment, promoted differentiation events. However, pathological hTSPCs showed a different expression pattern of tendon-related genes throughout the culture and an impaired balance of pro-inflammatory and anti-inflammatory cytokines, compared to healthy hTSPCs, as indicated by qRT-PCT and immunofluorescence analyses. Additionally, the expression of both tenogenic and cytokine genes in hTSPCs was influenced by hTGF-β1, indicating that the environment assembled was suitable for studying tendon stem cells differentiation. The study offers insights into the use of 3D cultures of hTSPCs as an in vitro model for investigating their behavior during tenogenic events and opens perspectives for following the potential impact on resident stem cells during regeneration and healing events

Tendon injury is debilitating and recalcitrant. With limited knowledge of disease aitiology we have are lacking in effective treatments for this prevalent musculoskeletal complaint. This presentation will outline our findings over the past few years in which we have demonstrated the importance of the interfascicular matrix (IFM) niche in maintaining healthy tendon function and driving disease progression. 1,2. It will also continue to describe our progress in developing both in vivo and in vitro models to interrogate disease progression. We have developed and validated a rat Achilles tendon overload model, in order to explore the impact of loading on IFM and fascicle structure, and the resulting cell response. Data highlights that structural disruption and inflammatory response both initiate in the IFM region, and can be seen in the absence of demonstrable changes to animal gait, indicating a sub-injury response in the tendon which we hypothesis may drive increased matrix turnover and repair. 3. . We are now looking to interrogate the pathways driving this inflammatory behaviour in an organ-chip model, exploring the interplay between IFM cells and cells within fascicles. We have demonstrated phenotypic distinction of cells from the two niche environments, localized the progenitor phenotype to the IFM region and demonstrated significant mechanosensitivity in the IFM cell population. 4. We are currently building appropriate niche environments to maintain cell phenotype in our in vitro models, to explore the metabolic changes associated with disease progression. Acknowledgements: This body of work has received funding from: BBSRC (BB/K008412 /1); Versus Arthritis (project grant 20262); Horserace Betting Levy Board (T5); Dunhill Medical Charity (project grant RPGF1802\23); MRC (MR/T015462/1)

Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by flow cytometry to obtain unbiased data sets on contained cell populations. Collagen from rat tail and human fibrin was used to facilitate a 3D culture environment for the BM-MNCs over a period of three days. Effects on cellular composition that could improve the regenerative potential of BM-MNCs within the BM autograft were assessed using flow cytometry. Cell-cell-interactions were visualized using confocal microscopy over a period of 24 hours. Cell localization and interaction partners were characterized using immunofluorescence labeled paraffin sectioning. Main BM-MNC populations like Monocytes, Macrophages, T cells and endothelial progenitor cells were determined and could be conserved in 3D culture over a period of three days. The 3D cultures will be further treated with already clinically available reagents that lead to effects even within a short-term exposure to stimulate angiogenic, osteogenic or immunomodulatory properties. These measures will help to ease the translation from “bench to bedside” into an intraoperative protocol in the end

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source