For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

Osteoarthritis (OA) is a degenerative disease that lacks regenerative treatment options. Current research focuses on mesenchymal stem cells (MSCs) and Platelet-Rich Plasma (PRP) as regenerative therapies, but extracellular vesicles (EVs) have shown to be more advantageous. This study compares the regenerative potential of human umbilical cord MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in ex vivo and in vivo OA models. In the ex vivo study, OA conditions were induced in human cartilage explants, which were then treated either with pEVs or cEVs. Results showed a higher content of DNA and collagen in the pEVs group compared to control and cEVs groups, suggesting that pEVs could be a potential alternative to cEVs. In the in vivo study, an OA model was established in the knee joints of rats through MIA (monoiodoacetate) injection and then treated either with pEVs or cEVs. Results showed that pEVs-treated knee joints had better subchondral bone integrity and greater OA reversion, particularly in female rats, indicating that pEVs are a viable regeneration treatment for OA and outperform cEVs in terms of efficacy. Overall, the study demonstrates the potential of EVs as a regenerative treatment for OA, with pEVs showing promising results in both ex vivo and in vivo models. The use of pEVs in clinical practice could provide a faster path to translation due to the established use of platelet concentrates in therapeutics. However, further studies are needed to fully evaluate the potential of pEVs for OA treatment and to elucidate the mechanisms behind their regenerative effects. Acknowledgments: The authors thank Dr Fernando Hierro (UIB) for their technical contribution with TEM, Mª Trinidad García (UIB) for the access to radioactivity facilities, Aina Arbós (IUNICS) for her contribution in the histology staining, María Tortosa (IdISBa) for her assistance with the animal care and ADEMA School of Dentistry for the access to the cone beam computed tomography (CBCT). Funding: This research was funded by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124), PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; the Direcció General d'Investigació and Conselleria d'Investigació, Govern Balear (FPI/2046/2017); the Mecanisme de Recuperació i Resiliència, intended to execute research projects of «Noves polítiques públiques per a un mercat de treball dinàmic, resilient i inclusiu», collected in Pla de Recuperació, Transformació i Resiliència, financed by European Union-Next Generation EU and driven by SOIB and Conselleria de Fons Europeus, Universitat i Cultura i la Conselleria de Model Econòmic, Turisme i Treball (NG0421) and the grant SYN20/03 from IdISBa

Introduction and Objective. Bone remodelling is a continuous process whereby osteocytes regulate the activity of osteoblasts and osteoclasts to repair loading-induced microdamage. While many in vitro studies have established the role of paracrine factors (e.g., RANKL/OPG) and cellular pathways involved in bone homeostasis, these techniques are generally limited to two-dimensional cell culture, which neglects the role of the native extracellular matrix in maintaining the phenotype of osteocyte. Recently, ex vivo models have been used to understand cell physiology and mechanobiology in the presence of the native matrix. Such approaches could be applicable to study the mechanisms of bone repair, whilst also enabling exploration of biomechanical cues. However, to date an ex vivo model of bone remodelling in cortical bone has not been developed. In this study, the objective was to develop an ex vivo model where cortical bone was subjected to cyclic strains to study the remodelling of bone. Materials and Methods. Ex vivo model of bone remodelling induced by cyclic loading: At the day of culling, beam-shape bovine bone samples were cut and preserved in PBS + 5% Pen/Strep + 2 mM L-Glut overnight at 37°C. Cyclic strains were applied with a three-point bend system to induce damage with a regime at 16.66 mm/min for 5,000 cycles in sterile PBS in Evolve® bags (maximum strain 6%). A control group was cultured under static conditions. Metabolic activity: Alamar Blue assays were performed after 1 and 7 days of ex vivo culture for each group (Static, Loaded) and normalized to weight. Bone remodelling: ALP activity was assessed in the media at day 1 and 7. After 24 hours cell culture conditioned media (CM) was collected from each group and stored at −80°C. RAW264.7 cells were cultured with CM for 6 days, after which the samples were stained for TRAP, to determine osteoclastogenesis, and imaged. Histomorphometry: Samples were cultured with calcein for 3 days to label bone formation between day 4 and 7. Fluorescent images were captured at day 7. μCT scanning was performed at 3 μm resolution after labelling samples with BaSO. 4. precipitate to quantify bone damage. Results. Bone was sectioned and cultured to maintain live osteoblasts and osteocytes. CM that was obtained 24 hours after cyclic loading and added to RAW264.7 cells cultures, resulted in significantly increased osteoclastogenic potential compared to that from static samples (4.245±1.65% vs 0.88±0.48%, p<0.001). Calcein and HE staining indicated the presence of structures similar to bone remodelling cones in both groups after 7 days of culture. Also, 7 days post-loading, matrix microdamage in the stimulated area, detected with the BaSO. 4. precipitate, were not significantly increased under the load point in loaded samples (0.11±0.05% of bone volume), while at the support areas it was significantly higher (0.2387±0.06%, p<0.001) compared to the static (0.062±0.02%). Conclusions. This study demonstrates that (1) cyclic strains applied on ex vivo bovine cortical bone successfully induced remodelling as characterized by the formation of bone resorption cones, along with an increase of osteoclast formation, and (2) there was an induction of microdamage post loading as shown by the significant increases in microdamage labelled. This supports previous in vivo studies with an increase in osteoclastogenesis up to 7 days post loading. This is the first evidence of the development of an ex vivo model to study osteon remodelling that could be applied to study bone physiology and repair

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation. Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion. The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin. The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo. Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

Cartilage diseases have a significant impact on the patient's quality of life and are a heavy burden for the healthcare system. Better understanding, early detection and proper follow-up could improve quality of life and reduce healthcare related costs. Therefore, the aim of this study was to evaluate if difference between osteoarthritic (OA) and non-osteoarthritic (non-OA) knees can be detected quantitatively on cartilage and subchondral bone levels with advanced but clinical available imaging techniques. Two OA (mean age = 88.3 years) and three non-OA (mean age = 51.0 years) human cadaveric knees were scanned two times. A high-resolution peripheral quantitative computed tomography (HR-pQCT) scan (XtremeCT, Scanco Medical AG, Switzerland) was performed to quantify the bone microstructure. A contrast-enhanced clinical CT scan (GE Revolution Evo, GE Medical Systems AG, Switzerland) was acquired with the contrast agent Visipaque 320 (60 ml) to measure cartilage. Subregions dividing the condyle in four parts were identified semi-automatically and the images were segmented using adaptive thresholding. Microstructural parameters of subchondral bone and cartilage thickness were quantified. The overall cartilage thickness was reduced by 0.27 mm between the OA and non-OA knees and the subchondral bone quality decreased accordingly (reduction of 33.52 % in BV/TV in the layer from 3 to 8 mm below the cartilage) for the femoral medial condyle. The largest differences were observed at the medial part of the femoral medial condyle both for cartilage and for bone parameters, corresponding to clinical observations. Subchondral bone microstructural parameters and cartilage thickness were quantified using in vivo available imaging and apparent differences between the OA and non-OA knees were detected. Those results may improve OA follow-up and diagnosis and could lead to a better understanding of OA. However, further in vivo studies are needed to validate these methods in clinical practice

A novel ex vivo intervertebral disc (IVD) organ model and corresponding sample holder were developed according to the requirements for six degrees of freedom loading and sterile culture in a new generation of multiaxial bioreactors. We tested if the model can be maintained in long-term IVD organ culture and validated the mechanical resistance of the IVD holder in compression, tension, torsion, and bending. An ex vivo bovine caudal IVD organ model was adapted by retaining 5-6 mm of vertebral bone to machine a central cross and a hole for nutrient access through the cartilaginous endplate. A counter cross was made on a customized, circular IVD holder. The new model was compared to a standard model with a minimum of bone for the cell viability and height changes after 3 weeks of cyclic compressive uniaxial loading (0.02-0.2 MPa, 0.2 Hz, 2h/ day; n= 3 for day 0, n= 2 for week 1, 2, and 3 endpoints). Mechanical tests were conducted on the assembly of IVD and holder enhanced with different combinations of side screws, top screws, and bone adhesive (n=3 for each test). The new model retained a high level of cell viability after three weeks of in vitro culture (outer annulus fibrosus 82%, inner annulus fibrosus 69%, nucleus pulposus 75%) and maintained the typical values of IVD height reduction after loading (≤ 10%). The holder-IVD interface reached the following highest average values in the tested configurations: 320.37 N in compression, 431.86 N in tension, 1.64 Nm in torsion, and 0.79 Nm in bending. The new IVD organ model can be maintained in long-term culture and when combined with the corresponding holder resists sufficient loads to study IVD degeneration and therapies in a new generation of multiaxial bioreactors

An ex vivo biomechanical test model for evaluating a novel bone adhesive has been developed. However, at day 1 in the in vivo pilot, high blood flow forced the study to halt until the solution presented here was developed. The profuse bleeding after bone core removal affected the bond strength and was reflected in the lower mean peak value 1.53N. After considering several options, we were successful in sealing the source of blood flow by pressing adhesive into place after bone core removal. After the initial adhesive had cured additional adhesive was used to secure the bone core in place. The animals were sacrificed after 24 h and a tensile test was undertaken on the bone core to failure. The ex vivo study produced mean peak tensile loads of 7.63N SD 2.39N (n=8, 4 rats 8 femurs). Whilst the mean peak tensile loads in the day 1 in vivo pilot were significantly lower 1.53N SD1.57 (n=8, 6 rats 8 femurs − 4 used for other tests). The subsequent layered adhesive bone cores showed a mean peak tensile force of 6.79N SD =3.13 (n=8, 4 rats 8 femurs). 7/8 failed at the bone to glue interface. This is the first successful demonstration of bonding bone in vivo for this class of adhesives. The development of a double adhesive method of fixing a bone core in the distal femur enabled mean peak tensile forces to be achieved in vivo at 24 hours that were comparable with the ex vivo results previously demonstrated. This method supports application in further animal series and over longer time scales. Biomaterials researchers that intend to use gel or paste like preparations in distal femur defects in the rat should be aware of the risks of biomaterial displacement by local blood flow

Articular cartilage has poor repair potential and the tissue formed is mechanically incompetent. Mesenchymal stromal cells (MSCs) show chondrogenic properties and the ability to re-grow cartilage, however a viable human model for testing cartilage regeneration and repair is lacking. Here, we describe an ex vivo pre-clinical femoral head model for studying human cartilage repair using MSCs. Human femoral heads (FHs) were obtained following femoral neck fracture with ethical permission/patient consent and full-depth cartilage wells made using a 3mm biopsy punch. Pancreas-derived mesenchymal stromal cells (P-MSC) were prepared in culture media at ~5000 cells/20µl and added to each well and leakage prevented with fibrin sealant. After 24hrs, the sealant was removed and medium replaced with StemPro. TM. chondrogenesis differentiation medium. The FHs were incubated (37. o. C;5% CO. 2. ) for 3wks, followed by a further 3wks in standard medium with 10% human serum with regular medium changes throughout. Compared to wells with medium only, A-MSCs produced a thin film across the wells which was excised en-block, fixed with 4% paraformaldehyde and frozen for cryo-sectioning. The cell/tissue films varied in thickness ranging over 20-440µm (82±21µm; mean±SEM; N=3 FHs). The thickness of MSC films abutting the cartilage wells was variable but generally greater (15-1880µm) than across the wells, suggesting an attachment to native articular cartilage. Staining of the films using safranin O (for glycosaminoglycans; quantified using ImageJ) was variable (3±8%; mean±SEM; N=3) but in one experiment reached 20% of the adjacent cartilage. A preliminary assessment of the repair tissue gave an O'Driscoll score of 10/24 (24 is best). These preliminary results suggest the ex vivo femoral head model has promise for studying the capacity of MSCs to repair cartilage directly in human tissue, although optimising MSCs to produce hyaline-like tissue is essential. Supported by the CSO (TCS/17/32)

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. (shared first authorship). Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1

Abstract. Objectives. Spinal disorders such as back pain incur a substantial societal and economic burden. Unfortunately, there is lack of understanding and treatment of these disorders are further impeded by the inability to assess spinal forces in vivo. The aim of this project is to address this challenge by developing and testing a novel image-driven approach that will assess the forces in an individual's spine in vivo by incorporating information acquired from multimodal imaging (magnetic resonance imaging (MRI) and biplane X-rays) in a subject-specific model. Methods. Magnetic resonance and biplane X-ray imaging are used to capture information about the anatomy, tissues, and motion of an individual's spine as they perform a range of everyday activities. This information is then utilised in a subject-specific computational model based on the finite element method to predict the forces in their spine. The project is also utilising novel machine learning algorithms and in vitro, six-axis mechanical testing on human, porcine and bovine samples to develop and test the modelling methods rigorously. Results & Discussion. MRI sequences have been identified that provide high-quality image data and information on different tissue types which will be used to predict subject-specific disc properties. In-vivo protocols to capture motion analysis, EMG muscle activity, and video X-rays of the spine have been designed with planned data collection of 15 healthy volunteers. Preliminary modelling work has evaluated potential machine learning approaches and quantified the sensitivity of the models developed to material properties. Conclusion. The development and testing of these image-driven subject-specific spine models will provide a new tool for determining forces in the spine. It will also provide new tools for measuring and modelling spine movement and quantifying the properties of the spinal tissues. Acknowledgments. Funding from the EPSRC: EP/V036602/1 (Meakin, Holsgrove & Javadi) and EP/V032275/1 (Holt & Williams). 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

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. Materials and Methods. We employed a previously established wedge defect model at the sheep tibia. The model incorporates two partial osteotomies directed perpendicularly to each other, thus creating a bone fragment in the shape of a wedge. The defect was instrumented with an active fixator that tilts the wedge around its apex to create a gradient of interfragmentary strain along the cutting line. The active fixator was equipped with a force and displacement sensors to measure the stiffness of the repair tissue during the course of healing. We developed a controller that enabled programming of different stimulation protocols and their autonomous execution during the in vivo experiment. The system was implanted in two sheep for a period of five weeks. The device was configured to execute immediate stimulation for one animal (stimulation from Day 1), and delayed stimulation for the other (stimulation from Day 22). The daily stimulation protocol consisted of 1’000 loading events evenly distributed over 12 hours from 9:00 am to 9:00 pm. The healing progression was monitored by the in vivo stiffness measurements provided by the fixator and by weekly radiographs. The impact of the local strain magnitude on bone formation was qualitatively evaluated on a post-mortem high-resolution CT scan of the animal with immediate stimulation. Results. The animals tolerated the fixator system well. Both devices operated seamlessly throughout the entire experiment. Callus formation was initiated earlier for the immediately stimulated animal which was also confirmed by a faster stiffness increase. In this pilot feasibility experiment, the initiation of callus formation was observed between 0% and 4% local interfragmentary strain. Conclusions. We developed an autonomous stimulation system for large animal research that enables systematic investigation of fracture healing processes. The in vivo pilot study demonstrated the feasibility of the system and delivered first interesting insides on temporal stimulation impact and callus induction strain limit. These observations, however, require further validation

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells. Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysis. Gram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats. This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration

Abstract. Objectives. Tendon and ligament injury poses an increasingly large burden to society. With surgical repair and grafting susceptible to high failure rates, tissue engineering provides novel avenues for treatment. This systematic review explores in vivo evidence whether mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can facilitate tendon and ligament repair in animal models. Methods. On May 26th 2021, a systematic search was performed on PubMed, Web of Science, Cochrane Library, Embase, using search terms ‘mesenchymal stem cell’ or ‘multipotent stem cell’ AND ‘extracellular vesicles’ or ‘exosomes’ AND ‘tendon’ or ‘ligament’ or ‘connective tissue’. Risk of bias was assessed using SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) tool. Studies administering EVs isolated from human or animal-derived MSCs into in vivo models of tendon/ligament injury were included. In vitro, ex vivo, in silico studies were excluded, and studies without a control group were excluded. Data on isolation and characterisation of MSCs and EVs, and in vivo findings in animal models were extracted. Results. Out of 383 relevant studies, 11 case-control studies were included for data extraction, including a total of 448 animal subjects (range 10–90). Six studies utilised bone marrow-derived MSCs. All studies characterised their MSCs via flow cytometry, which expressed CD44 and CD90, and isolated EVs via ultracentrifugation (average diameter 125nm). Five studies utilised histological scoring systems, all of which reported a lower score with EV treatment, suggesting improved healing ability. Four studies reported increased anti-inflammatory cytokine expression (IL-10, TGF-β1); three studies reported decreased endogenous M1/M2 macrophage ratio with EV treatment. Eight studies reported increased maximum stiffness, breaking load, tensile strength in EV-treated tendons. Conclusion. MSC-EVs are effective therapeutic agents for tendon/ligament pathologies, attenuating the initial inflammatory response, and accelerating tendon matrix regeneration. Future randomised controlled trials are needed to definitely demonstrate MSC-EVs superiority in management of tendon/ligament injury

Total knee replacement (TKR) design aims to restore normal kinematics with emphasis on flexion range. The survivorship of a TKR is dependent on the kinematics in six-degrees-of-freedom (6-DoF). Stepping up, such as stair ascent is a kinematically demanding activity after TKR. The debate about design choice has not yet been informed by 6-DoF in vivo kinematics. This prospective randomised controlled trial (RCT) compared kneeling kinematics in three TKR designs. 68 participants were randomised to receive either cruciate retaining (CR-FB), rotating platform (CR-RP) or posterior stabilised (PS-FB) prostheses. Image quality was sufficient for 49 of these patients to be included in the final analysis following a minimum 1-year follow-up. Patients completed a step-up task while being imaged using single-plane fluoroscopy. Femoral and tibial computer-aided design (CAD) models for each of the TKR designs were registered to the fluoroscopic images using bespoke software OrthoVis to generate six-degree-of-freedom kinematics. Differences in kinematics between designs were compared as a function of flexion. There were no differences in terminal extension between the groups. The CR-FB was further posterior and the CR-RP was more externally rotated at terminal extension compared to the other designs. Furthermore, the CR-FB designs was more posteriorly positioned at each flexion angle compared to both other designs. Additionally, the CR-RP design had more external femoral rotation throughout flexion when compared with both fixed bearing designs. However, there were no differences in total rotation for either step-up or down. Visually, it appears there was substantial variability between participants in each group, indicating unique patient-specific movement patterns. While use of a specific implant design does influence some kinematic parameters, the overall patterns are similar. Furthermore, there is high variability indicating patient-specific kinematic patterns. At a group level, none of these designs appear to provide markedly different step-up kinematic patterns. This is important for patient expectations following surgery. Future work should aim to better understand the unique patient variability

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2

Objectives. This systematic review aimed to assess the in vivo and clinical effect of strontium (Sr)-enriched biomaterials in bone formation and/or remodelling. Methods. A systematic search was performed in Pubmed, followed by a two-step selection process. We included in vivo original studies on Sr-containing biomaterials used for bone support or regeneration, comparing at least two groups that only differ in Sr addition in the experimental group. Results. A total of 572 references were retrieved and 27 were included. Animal models were used in 26 articles, and one article described a human study. Osteoporotic models were included in 11 papers. All articles showed similar or increased effect of Sr in bone formation and/or regeneration, in both healthy and osteoporotic models. No study found a decreased effect. Adverse effects were assessed in 17 articles, 13 on local and four on systemic adverse effects. From these, only one reported a systemic impact from Sr addition. Data on gene and/or protein expression were available from seven studies. Conclusions. This review showed the safety and effectiveness of Sr-enriched biomaterials for stimulating bone formation and remodelling in animal models. The effect seems to increase over time and is impacted by the concentration used. However, included studies present a wide range of study methods. Future work should focus on consistent models and guidelines when developing a future clinical application of this element. Cite this article: N. Neves, D. Linhares, G. Costa, C. C. Ribeiro, M. A. Barbosa. In vivo and clinical application of strontium-enriched biomaterials for bone regeneration: A systematic review. Bone Joint Res 2017;6:366–375. DOI: 10.1302/2046-3758.66.BJR-2016-0311.R1

Abstract. Objective. Articular cartilage damaged through trauma or disease has a limited ability to repair. Untreated, these focal lesions progress to generalized changes including osteoarthritis. Musculoskeletal disorders including osteoarthritis are the most significant contributor to disability globally. There is increasing interest in the use of mesenchymal stem cells (MSCs) for the treatment of focal chondral lesions. There is some evidence to suggest that the tissue type from which MSCs are harvested play a role in determining their ability to regenerate cartilage in vitro and in vivo. In humans, MSCs derived from synovial tissue may have superior chondrogenic potential. Methods. We carried out a systematic literature review on the effectiveness of synovium-derived MSCs (sMSCs) in cartilage regeneration in in vivo studies in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. Nineteen studies were included in our review; four examined the use of human sMSCs and the remainder were conducted using sMSCs harvested from animals. Results. Despite the variability of animals, cell harvesting techniques, methods of delivery, and outcome measures, all studies reported successful cartilage repair with sMSC transplantation. Conclusion. We conclude that sMSC transplantation holds promise as a treatment option for focal cartilage defects. We believe that defining the cell population being used, establishing standardized methods for MSC delivery, and the use of objective outcome measures should enable future high-quality studies such as randomized controlled clinical trials to provide the evidence needed to manage chondral lesions optimally. 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

Engineered cartilage is poorly organized and fails to recapitulate physiologic organization in a hyaline upper and a mineralizing bottom zone deemed important for proper function. Objective was to grow bizonal human cartilage constructs in which in vivo mineralization is self-restricted to the bottom zone. Self-assembling biomaterial-free cell discs were generated from mesenchymal stroma cells and allowed to accumulate proteoglycans and collagen-type II over 3 weeks. In vitro mineralization of the cell discs with four mineralization media for up to 8 weeks showed that calcification was supported in all media containing ß-glycerophosphate. However, proteoglycans were retained only in media containing insulin. Bizonal cartilage constructs were made from 3-week non-mineralized cell discs overlaid with chondrocyte-seeded starPEG-heparin hydrogel or with a fibrin-gel layer to select the best design for upper zone development. Freshly prepared zonal constructs were implanted into subcutaneous pouches of immuno-deficient mice to compare in vivo development. After 6 weeks in vivo, both construct types were rich in collagen-type II in the upper zone and contained a mineralized bottom zone. However, solely for starPEG constructs, tissue volume of the upper zone remained high and alkaline phosphatase, alizarin red, and collagen-type X staining were restricted to the bottom zone. StarPEG zonal constructs were superior to fibrin constructs due to self-restriction of mineralization and hypertrophic markers to the bottom zone. This innovative design of bizonal constructs offers the successful generation of an organized cartilage resembling the native cartilage with the chance for immediate use of autogenous chondrocytes in a one-step surgical joint intervention