R399E, A Mutated Form of Growth and Differentiation Factor 5, for Disease Modification of Osteoarthritis.

OBJECTIVE: To preclinically characterize a mutant form of growth and differentiation factor 5, R399E, with reduced osteogenic properties as a potential disease-modifying osteoarthritis (OA) drug. METHODS: Cartilage, synovium, and meniscus samples from patients with OA were used to evaluate anabolic and antiinflammatory properties of R399E. In the rabbit joint instability model, 65 rabbits underwent transection of the anterior cruciate ligament plus partial meniscectomy. Three intraarticular (IA) R399E doses were administered biweekly 6 times, and static incapacitance was determined to assess joint pain. OA was evaluated 13 weeks after surgery. In sheep, medial meniscus transection was performed to induce OA, dynamic weight bearing was measured in-life, and OA was assessed after 13 weeks. RESULTS: Intermittent exposure to R399E (1 week per month) was sufficient to induce cell proliferation and release of anabolic markers in 3-dimensional chondrocyte cultures. R399E also inhibited the release of interleukin-1ß (IL-1ß), IL-6, and prostaglandin E2 from cartilage with synovium, meniscal cell, and synoviocyte cultures. In rabbits, the mean difference (95% confidence interval [95% CI]) in weight bearing for R399E compared to vehicle was -5.8 (95% confidence interval [95% CI] -9.54, -2.15), -7.2 (95% CI -10.93, -3.54), and -7.7 (95% CI -11.49, -3.84) for the 0.6, 6, and 60 µg doses, respectively, 6 hours after the first IA injection, and was statistically significant through the entire study for all doses. Cartilage surface structure improved with the 6-µg dose. Structural and symptomatic improvement with the same dose was confirmed in the sheep model of OA. CONCLUSION: R399E influences several pathologic processes contributing to OA, highlighting its potential as a disease-modifying therapy.

Role of Human Mesenchymal Stem Cells and Derived Extracellular Vesicles in Reducing Sensory Neuron Hyperexcitability and Pain Behaviors in Murine Osteoarthritis.

OBJECTIVE: Mesenchymal stem/stromal cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) have been reported to alleviate pain in patients with knee osteoarthritis (OA). We undertook this study to determine whether MSCs and/or MSC-EVs reduce OA pain through influencing sensory neuron excitability in OA joints. METHODS: We induced knee OA in adult male C57BL/6J mice through destabilization of the medial meniscus (DMM) surgery. Mice were sorted into 4 experimental groups with 9 mice per group as follows: unoperated sham, untreated DMM, DMM plus MSC treatment, and DMM plus MSC-EV treatment. Treated mice received either MSCs at week 14 postsurgery or MSC-EVs at weeks 12 and 14 postsurgery. Mouse behavior was evaluated by digging and rotarod tests and the Digital Ventilated Cage system. At week 16, mouse knee joints were harvested for histology, and dorsal root ganglion (DRG) neurons were isolated for electrophysiology. Furthermore, we induced hyperexcitability in DRG neurons in vitro using nerve growth factor (NGF) then treated these neurons with or without MSC-EVs and evaluated neuron excitability. RESULTS: MSC- and MSC-EV-treated DMM-operated mice did not display pain-related behavior changes (in locomotion, digging, and sleep) that occurred in untreated DMM-operated mice. The absence of pain-related behaviors in MSC- and MSC-EV-treated mice was not the result of reduced joint damage but rather a lack of knee-innervating sensory neuron hyperexcitability that was observed in untreated DMM-operated mice. Furthermore, we found that NGF-induced sensory neuron hyperexcitability is prevented by MSC-EV treatment (P < 0.05 versus untreated NGF-sensitized neurons when comparing action potential threshold). CONCLUSION: MSCs and MSC-EVs may reduce pain in OA by direct action on peripheral sensory neurons.

Sustained delivery of the bone morphogenetic proteins BMP-2 and BMP-7 for cartilage repair and regeneration in osteoarthritis.

Objective: Attempts to utilise growth factors (GF) such as bone morphogenic proteins (BMPs) to treat osteoarthritis (OA) in the clinic have not secured widespread adoption. However, the novel crystalline GF formulation called PODS offers new perspectives. This study investigated the hypothesis that Polyhedrin Delivery System (PODS) BMP-2 and PODS BMP-7, compared with conventional BMP-2 and BMP-7 increase capacity for cartilage repair. Design: Sustained release from PODS BMP-2 and PODS BMP-7 and their effects on OA patient-derived cells as well as a chondrocyte cell line were first assessed in vitro. Here, extra cellular matrix (ECM) protein gene expression and actual ECM deposition were measured and compared to the effect achieved with conventional, soluble BMPs. Subsequently, in an established murine model of cartilage regeneration of the knee joint, changes were traced over 8 weeks and scored with two metrics, modified Pineda and Mankin. Results: Both crystalline PODS BMP formulations strongly induced proliferation in primary as well as immortal cell line chondrocytes, outperforming conventional soluble BMP-2 and BMP-7. Furthermore, ECM-producing genes were upregulated and the production of ECM could be demonstrated. In the murine cartilage regeneration model, both PODS BMP-2 and PODS-BMP-7 improved cartilage repair assessed with both histological scoring methods. Conclusions: This study showed that the sustained release of GF from PODS BMPs is effective in promoting chondrogenesis in vitro. The small animal data suggests that this novel approach of delivering therapeutic proteins sustainably and locally to the knee has promise for developing future disease-modifying therapies of OA.

The anti-inflammatory effects of equine bone marrow stem cell-derived extracellular vesicles on autologous chondrocytes.

BACKGROUND: Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. OBJECTIVES: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. STUDY DESIGN: An in vitro assessment of the effect of direct culturing extracellular vesicles on artificially inflamed chondrocytes. METHODS: Extracellular vesicles were isolated from bone marrow MSC using differential sequential ultracentrifugation. Vesicles were characterised using electron microscopy, nanoparticle tracing analysis and protein analysis. Vesicle internalisation was carried out via vesicles being pre-stained and co-cultured with equine chondrocytes before analysis using confocal microscopy. The effects of vesicles on artificially inflamed chondrocytes was examined using quantitative PCR. RESULTS: To the best of the authors' knowledge, this is the first study to isolate and characterise extracellular vesicles from equine bone MSC. Vesicles were taken up by autologous chondrocytes and had anti-inflammatory effects on gene expression following chondrocyte exposure to tumour necrosis factor α and Interleukin 1ß. MAIN LIMITATIONS: Only three independent biological repeats were performed and the work was done in vitro. CONCLUSION: Extracellular vesicles can be isolated from equine bone marrow MSC; they may be taken up by chondrocytes and have an anti-inflammatory action.

Use of autologous products for the treatment of joint and soft tissue disease in horses: A systematic review.

BACKGROUND: Soft tissue injuries and joint disease are the predominate causes of lameness in the equine athlete and these pathologies carry a guarded prognosis for a return to previous performance. Recently the use of autologous products has become more widespread as a treatment in equine sports medicine. However, the efficacy of these products is yet to be fully established. OBJECTIVE: To evaluate the current published evidence base regarding the efficacy of autologous products in soft tissue injuries and joint disease. METHODS: A systematic review of English articles using MEDLINE, EMBASE and Web of Science databases from 1980 to 2017. The search strategy identified 1594 papers for review. RESULTS: Fifty-eight papers were included in this review, 28 of which were randomised controlled trials. Significant benefit was reported under several parameters, most notably in the use of autologous chondrocytes in artificially induced cartilage defects on histology. One paper documented a significant clinical response under lameness examination. CONCLUSION: The current literature shows that the treatment of soft tissue injury and cartilage disease with autologous products is safe and that the use of some products can give significant benefit on some outcome measures. True clinical significance is yet to be demonstrated with any product.

Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect.

Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis (OA) and cause of debilitating pain in patients. Rodent OD models are less translatable because of their smaller joint size and open growth plate. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. A unilateral 6-mm deep OD was induced in adult female sheep. Two to six weeks after operation, lumbar dorsal root ganglia (DRG) neurons were collected from the contralateral (Ctrl) and OD side of operated sheep. Functional assessment of neuronal excitability and activity of the pain-related ion channels transient receptor potential vanilloid receptor 1 (TRPV1) and P2X3 was conducted using electrophysiology and Ca2+ imaging. Immunohistochemistry was used to verify expression of pain-related proteins. We observed that an increased proportion of OD DRG neurons (sheep, N = 3; Ctrl neurons, n = 15, OD neurons, n = 16) showed spontaneous electrical excitability (Ctrl: 20.33 ± 4.5%; OD: 50 ± 10%; p = 0.009, unpaired t test) and an increased proportion fired a greater number of spikes above baseline in response to application of a TRPV1 agonist (capsaicin) application (Ctrl: 40%; OD: 75%; p = 0.04, χ2 test). Capsaicin also produced Ca2+ influx in an increased proportion of isolated OD DRG neurons (Ctrl: 25%; OD: 44%; p = 0.001, χ2 test). Neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Overall, we provide evidence of increased excitability of DRG neurons (an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.

Using apheresis-derived cells to augment microdrilling in the treatment of chondral defects in an ovine model.

The treatment of chondral defects using microdrilling often results in a mechanically weak fibrocartilagenous repair, rather than a more robust hyaline cartilage repair. Many different microfracture/microdrilling augmentation techniques have been described, including the use of cellular products to enhance healing. Autologous peripheral blood progenitor cells can be obtained via apheresis after administration of granulocyte colony-stimulating factor (G-CSF) and have been used successfully to augment microdrilling in clinical patients. The objective of this study was to use apheresis-derived mononuclear blood cells to augment microdrilling treatment of a cartilage defect in an ovine model to determine the effect on healing. Forty adult female sheep were used in this study and were divided into a control group (microdrilling alone) and a treatment group (microdrilling, hyaluronic acid, and apheretic product). Outcome measurements included weight-bearing on the operated limb, macroscopic scoring of the joint, histology, and immunohistochemistry. In addition, magnetic resonance imaging was used to attempt to identify SPION-labeled cells from the apheretic product in the operated limbs. The results showed a significant increase in healing as measured by the modified O'Driscoll sore in the treated group. No evidence of homing of SPION-labeled cells to the defect was found and no correlation was found between the response to G-CSF administration or concentration of CD34+  and outcome. A correlation was found between healing and the concentration of white blood cells and peripheral blood mononuclear cell numbers in the apheretic product.

The optimisation of deep neural networks for segmenting multiple knee joint tissues from MRIs.

Automated semantic segmentation of multiple knee joint tissues is desirable to allow faster and more reliable analysis of large datasets and to enable further downstream processing e.g. automated diagnosis. In this work, we evaluate the use of conditional Generative Adversarial Networks (cGANs) as a robust and potentially improved method for semantic segmentation compared to other extensively used convolutional neural network, such as the U-Net. As cGANs have not yet been widely explored for semantic medical image segmentation, we analysed the effect of training with different objective functions and discriminator receptive field sizes on the segmentation performance of the cGAN. Additionally, we evaluated the possibility of using transfer learning to improve the segmentation accuracy. The networks were trained on i) the SKI10 dataset which comes from the MICCAI grand challenge "Segmentation of Knee Images 2010″, ii) the OAI ZIB dataset containing femoral and tibial bone and cartilage segmentations of the Osteoarthritis Initiative cohort and iii) a small locally acquired dataset (Advanced MRI of Osteoarthritis (AMROA) study) consisting of 3D fat-saturated spoiled gradient recalled-echo knee MRIs with manual segmentations of the femoral, tibial and patellar bone and cartilage, as well as the cruciate ligaments and selected peri-articular muscles. The Sørensen-Dice Similarity Coefficient (DSC), volumetric overlap error (VOE) and average surface distance (ASD) were calculated for segmentation performance evaluation. DSC ≥ 0.95 were achieved for all segmented bone structures, DSC ≥ 0.83 for cartilage and muscle tissues and DSC of ≈0.66 were achieved for cruciate ligament segmentations with both cGAN and U-Net on the in-house AMROA dataset. Reducing the receptive field size of the cGAN discriminator network improved the networks segmentation performance and resulted in segmentation accuracies equivalent to those of the U-Net. Pretraining not only increased segmentation accuracy of a few knee joint tissues of the fine-tuned dataset, but also increased the network's capacity to preserve segmentation capabilities for the pretrained dataset. cGAN machine learning can generate automated semantic maps of multiple tissues within the knee joint which could increase the accuracy and efficiency for evaluating joint health.

Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis.

Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that agrin is a potent chondrogenic factor and that a single intraarticular administration of agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of ß-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of agrin for joint surface regeneration.

Peripheral mechanisms of arthritic pain: A proposal to leverage large animals for in vitro studies.

Pain arising from musculoskeletal disorders such as arthritis is one of the leading causes of disability. Whereas the past 20-years has seen an increase in targeted therapies for rheumatoid arthritis (RA), other arthritis conditions, especially osteoarthritis, remain poorly treated. Although modulation of central pain pathways occurs in chronic arthritis, multiple lines of evidence indicate that peripherally driven pain is important in arthritic pain. To understand the peripheral mechanisms of arthritic pain, various in vitro and in vivo models have been developed, largely in rodents. Although rodent models provide numerous advantages for studying arthritis pathogenesis and treatment, the anatomy and biomechanics of rodent joints differ considerably to those of humans. By contrast, the anatomy and biomechanics of joints in larger animals, such as dogs, show greater similarity to human joints and thus studying them can provide novel insight for arthritis research. The purpose of this article is firstly to review models of arthritis and behavioral outcomes commonly used in large animals. Secondly, we review the existing in vitro models and assays used to study arthritic pain, primarily in rodents, and discuss the potential for adopting these strategies, as well as likely limitations, in large animals. We believe that exploring peripheral mechanisms of arthritic pain in vitro in large animals has the potential to reduce the veterinary burden of arthritis in commonly afflicted species like dogs, as well as to improve translatability of pain research into the clinic.

Author Correction: Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Treatment of Cartilage Damage Using Human Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Systematic Review of in vivo Studies.

Damage to joints through injury or disease can result in cartilage loss, which if left untreated can lead to inflammation and ultimately osteoarthritis. There is currently no cure for osteoarthritis and management focusses on symptom control. End-stage osteoarthritis can be debilitating and ultimately requires joint replacement in order to maintain function. Therefore, there is growing interest in innovative therapies for cartilage repair. In this systematic literature review, we sought to explore the in vivo evidence for the use of human Mesenchymal Stem Cell-derived Extracellular Vesicles (MSC-EVs) for treating cartilage damage. We conducted a systematic literature review in accordance with the PRISMA protocol on the evidence for the treatment of cartilage damage using human MSC-EVs. Studies examining in vivo models of cartilage damage were included. A risk of bias analysis of the studies was conducted using the SYRCLE tool. Ten case-control studies were identified in our review, including a total of 159 murine subjects. MSC-EVs were harvested from a variety of human tissues. Five studies induced osteoarthritis, including cartilage loss through surgical joint destabilization, two studies directly created osteochondral lesions and three studies used collagenase to cause cartilage loss. All studies in this review reported reduced cartilage loss following treatment with MSC-EVs, and without significant complications. We conclude that transplantation of MSC-derived EVs into damaged cartilage can effectively reduce cartilage loss in murine models of cartilage injury. Additional randomized studies in animal models that recapitulates human osteoarthritis will be necessary in order to establish findings that inform clinical safety in humans.

Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model.

Multipotent Mesenchymal Stem/Stromal Cells (MSCs) are widely used in cellular therapy for joint repair. However, the use of MSC therapies is complicated by a lack of understanding of the behaviour of cells and repair within the joint. Current methods of MSC tracking include labelling the cells with Super Paramagnetic Iron Oxide nanoparticles (SPIOs). However, standard acquisition sequences (T2 and T2*) give poor anatomical definition in the presence of SPIOs. To avoid anatomical compromise in the presence of SPIOs, we have investigated the use of Ultra-short Echo Time (UTE) MRI, using a 3D cones acquisition trajectory. This method was used to track SPIO labelled MSC injected into joints containing osteochondral defects in experimental sheep. This study demonstrates that multiple echo times from UTE with 3 T MRI can provide excellent anatomical detail of osteochondral defects and demonstrate similar features to histology. This work also monitors the location of SPIO-labelled cells for regenerative medicine of the knee with MRI, histology, and Prussian blue staining. With these methods, we show that the SPIOs do not hone to the site of defect but instead aggregate in the location of injection, which suggests that any repair mechanism with this disease model must trigger a secondary process.

Early-Onset Osteoarthritis originates at the chondrocyte level in Hip Dysplasia.

Subjects with developmental dysplasia of the hip (DDH) often show early-onset osteoarthritis (OA); however, the molecular mechanisms underlying this pathology are not known. We investigated whether cellular changes in chondrocytes from OA cartilage can be detected in chondrocytes from DDH cartilage before histological manifestations of degeneration. We characterized undamaged and damaged articular cartilage from 22 participants having hip replacement surgery with and without DDH (9 DDH-OA, 12 OA-only, one femoral fracture). Tissue immunostaining revealed changes in damaged OA-only cartilage that was also found in undamaged DDH-OA cartilage. Chondrocytes in situ from both groups show: (i) thicker fibers of vimentin intermediate filaments, (ii) clusters of integrin α5ß1, (iii) positive MMP13 staining and (iv) a higher percentage of cells expressing the serine protease HtrA1. Further characterization of the extracellular matrix showed strong aggrecan and collagen II immunostaining in undamaged DDH cartilage, with no evidence of augmented cell death by activation of caspase 3. These findings suggest that early events in DDH cartilage originate at the chondrocyte level and that DDH cartilage may provide a novel opportunity to study these early changes for the development of therapeutic targets for OA.

Ex vivo MRI cell tracking of autologous mesenchymal stromal cells in an ovine osteochondral defect model.

BACKGROUND: Osteochondral injuries represent a significant clinical problem requiring novel cell-based therapies to restore function of the damaged joint with the use of mesenchymal stromal cells (MSCs) leading research efforts. Pre-clinical studies are fundamental in translating such therapies; however, technologies to minimally invasively assess in vivo cell fate are currently limited. We investigate the potential of a MRI- (magnetic resonance imaging) and superparamagnetic iron oxide nanoparticle (SPION)-based technique to monitor cellular bio-distribution in an ovine osteochondral model of acute and chronic injuries. METHODS: MSCs were isolated, expanded and labelled with Nanomag, a 250-nm SPION, and using a novel cell-penetrating technique, glycosaminoglycan-binding enhanced transduction (GET). MRI visibility thresholds, cellular toxicity and differentiation potential post-labelling were assessed in vitro. A single osteochondral defect was created in the medial femoral condyle in the left knee joint of each sheep with the contralateral joint serving as the control. Cells, either GET-Nanomag labelled or unlabelled, were delivered 1 week or 4.5 weeks later. Sheep were sacrificed 7 days post implantation and immediately MR imaged using a 0.2-T MRI scanner and validated on a 3-T MRI scanner prior to histological evaluation. RESULTS: MRI data demonstrated a significant increase in MRI contrast as a result of GET-Nanomag labelling whilst cell viability, proliferation and differentiation capabilities were not affected. MRI results revealed evidence of implanted cells within the synovial joint of the injured leg of the chronic model only with no signs of cell localisation to the defect site in either model. This was validated histologically determining the location of implanted cells in the synovium. Evidence of engulfment of Nanomag-labelled cells by leukocytes is observed in the injured legs of the chronic model only. Finally, serum c-reactive protein (CRP) levels were measured by ELISA with no obvious increase in CRP levels observed as a result of P21-8R:Nanomag delivery. CONCLUSION: This study has the potential to be a powerful translational tool with great implications in the clinical translation of stem cell-based therapies. Further, we have demonstrated the ability to obtain information linked to key biological events occurring post implantation, essential in designing therapies and selecting pre-clinical models.

Healing of Osteochondral Defects via Endochondral Ossification in an Ovine Model.

OBJECTIVE: The objective of this study was to describe the mechanism of healing of osteochondral defects of the distal femur in the sheep, a commonly used translational model. Information on the healing mechanism be useful to inform the design of tissue engineering devices for joint surface defect repair. DESIGN: A retrospective study was conducted examining 7-mm diameter osteochondral defects made in the distal medial femoral condyle of 40 adult female sheep, comprising control animals from 3 separate structures. The healing of the defects was studied at post mortem at up to 26 weeks. RESULTS: Osteochondral defects of the distal femur of the sheep heal through endochondral ossification as evidenced by chondrocyte hypertrophy and type X collagen expression. Neocartilage is first formed adjacent to damaged cartilage and then streams over the damaged underlying bone before filling the defect from the base upward. No intramembranous ossification or isolated mesenchymal stem cell aggregates were detected in the healing tissue. No osseous hypertrophy was detected in the defects. CONCLUSIONS: Osteochondral defects of the medial femoral condyle of the sheep heal via endochondral ossification, with neocartilage first appearing adjacent to damaged cartilage. Unlike the mechanism of healing in fracture repair, neocartilage is eventually formed directly onto damaged bone. There was most variability between animals between 8 and 12 weeks postsurgery. These results should be considered when designing devices to promote defect healing.

The regulation of sclerostin by cathepsin K in periodontal ligament cells.

Sclerostin is a clinically important protein with key functions in the musculoskeletal system playing a key role in bone formation and remodelling. Whilst a wide range of mechanisms have been identified which regulate sclerostin expression, little is known about the degradation of the protein. The aim of this study was to identify enzymes capable of degrading sclerostin in peridontal ligament (PDL) fibroblasts cells in vitro and to investigate the functionality of these enzymes. We have demonstrated that cathepsin K cleaves sclerostin in vitro in PDL. We have shown that cathepsin K and sclerostin are co-localised in PDL cells and, using cathepsin K knockdown experiments, we have shown that cathepsin K actively controls sclerostin levels in these cells, through a lysosomal mechanism that is affected by hypoxia. These results are the first description of the degradative control of sclerostin in musculoskeletally derived cells in vitro and suggest that degradation of the protein may well play an important role in the control of bone formation and remodelling.

Osteoblast differentiation of equine induced pluripotent stem cells.

Bone fractures occur in horses following traumatic and non-traumatic (bone overloading) events. They can be difficult to treat due to the need for the horse to bear weight on all legs during the healing period. Regenerative medicine to improve fracture union and recovery could significantly improve horse welfare. Equine induced pluripotent stem cells (iPSCs) have previously been derived. Here we show that equine iPSCs cultured for 21 days in osteogenic induction media on an OsteoAssay surface upregulate the expression of osteoblast associated genes and proteins, including COL1A1, SPARC, SPP1, IBSP, RUNX2 and BGALP We also demonstrate that iPSC-osteoblasts are able to produce a mineralised matrix with both calcium and hydroxyapatite deposition. Alkaline phosphatase activity is also significantly increased during osteoblast differentiation. Although the genetic background of the iPSC donor animal affects the level of differentiation observed after 21 days of differentiation, less variation between lines of iPSCs derived from the same horse was observed. The successful, direct, differentiation of equine iPSCs into osteoblasts may provide a source of cells for future regenerative medicine strategies to improve fracture repair in horses undergoing surgery. iPSC-derived osteoblasts will also provide a potential tool to study equine bone development and disease.

Peripheral mononuclear blood cell apheresis in a preclinical ovine model.

BACKGROUND: Recent research has demonstrated that circulating peripheral blood mononuclear fractions (PBMC) containing haematopoietic stem (HSC)/progenitor cells have the potential to play a crucial role in regenerative medicine strategies. Work in our laboratory has shown that a peripheral blood mononuclear cell fraction (PBMC) enhances cartilage repair in an osteochondral defect model in sheep and has a significant effect on cells in the joint niche. In order to obtain PBMC rich blood containing HSCs for further studies, we have performed, for the first time, apheresis on adult sheep. RESULTS: Subcutaneous granulocyte-colony stimulating factor (G-CSF) was used to mobilise white blood cells and continual flow apheresis was performed on 8 sheep under general anaesthetic. There were no observable side effects, although a marked tendency for blood clotting during the procedure was noted. The administration of G-CSF for 3 days increased the white blood cell (WBC) count in the peripheral blood from to 6.7 ± 2.1 × 106/ml to 16.1 ± 5.0 × 106/ml. Following apheresis, the WBC numbers in the apheretic product increased to 38.5 ± 27.6 × 106/ml, comprised of a significant increase in neutrophils and PBMC (from 5.25 ± 1.8 × 106/ml following G-CSF stimulation to 27.5 5 ± 27.6 × 106/ml). There was a mean of 2.1% CD34 + ve cells and 95.5% CD45 + ve cells in the apheretic product. CONCLUSIONS: This study describes the administration of G-CSF and subsequent apheresis in adult sheep. The technique is safe when performed as described with no observable side effects. The technique permits collection of an increased WBC fraction containing neutrophils and PBMC in adult sheep. This apheretic product contains CD34 + ve cells, representing an HSC/progenitor population for use in in vivo and in vitro experiments.

"Patient reported outcomes" following experimental surgery-using telemetry to assess movement in experimental ovine models.

Many potential treatments for orthopedic disease fail at the animal to human translational hurdle. One reason for this failure is that the majority of pre-clinical outcome measurements emphasize structural changes, such as gross morphology and histology, and do not address pain or its alleviation, which is a key component of treatment success in man. With increasing emphasis on "patient reported outcome measurements (PROM)" in clinical practice, in this study we have used two different telemetric methods (geolocation and Fitbark activity trackers, Kansas City, MO) to measure movement behavior, i.e., an indirect PROM, in an ovine osteoarthritis induction and an osteochondral defect model performed in adult female Welsh Mountain sheep. This study demonstrates that both systems can be used to track movement and activity of experimental sheep before and after surgery and that the Geolocator system recorded a decrease in distance moved and activity at the end of the experimental period in both models. The Fitbark activity tracker also recorded significant alterations in movement behavior at the end of these studies and this method of recording showed a correlation between Fitbark data and radiography, macroscopic and histological scoring (well recognized outcome measurements), particularly in animals with large (10 mm) defects, i.e., more severe pathology. These results suggest that telemetry is able to track movement behavior in experimental sheep and that the methodology should be considered for inclusion in outcome measures in preclinical orthopedic research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:1498-1507, 2018.