Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair - effect of BMP-2.

BACKGROUND: Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2) on this process was investigated. METHODS: Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. RESULTS: Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. CONCLUSION: These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Alterations in the vimentin cytoskeleton in response to single impact load in an in vitro model of cartilage damage in the rat.

BACKGROUND: Animal models have provided much information on molecular and cellular changes in joint disease, particularly OA. However there are limitations to in vivo work and single tissue in vitro studies can provide more specific information on individual events. The rat is a commonly used laboratory species but at the current time only in vivo models of rat OA are available to study. The purpose of this study was to investigate the damage that single impact load (SIL) of 0.16J causes in a rat cartilage in vitro model and assess whether this load alters the arrangement of vimentin. METHODS: Rat cartilage was single impact loaded (200 g from 8 cm) and cultured for up to 48 hours (n = 72 joints). Histological changes were measured using a semi-quantitative modified Mankin score. Immunolocalisation was used to identify changes in vimentin distribution. RESULTS: SIL caused damage in 32/36 cartilage samples. Damage included surface fibrillation, fissures, fragmentation, changes in cellularity and loss of proteoglycan. SIL caused a statistically significant increase in modified Mankin score and chondrocyte clusters over time. SIL caused vimentin disassembly (as evidenced by collapse of vimentin around the nucleus). CONCLUSION: This study describes a model of SIL damage to rat cartilage. SIL causes changes in histological/chemical parameters which have been measured using a semi-quantitative modified Mankin score. Single impact load also causes changes in the pattern of vimentin immunoreactivity, indicating vimentin dissassembley. Using a semi-quantitative scoring system the disassembly was shown to be statistically significant in SIL damaged cartilage. The changes described in this paper suggest that this novel single tissue rat model of joint damage is a possible candidate model to replace in vivo models.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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.Anne Duchess of Cambridge Charitable Trust, Paul Mellon Foundation, Cambridge Turs

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.European Union's Horizon 2020 Framework Programme [grant number 761214] Addenbrooke’s Charitable Trust (ACT) National Institute of Health Research (NIHR) Cambridge Biomedical Research Centre University of Cambridge Cambridge University Hospitals NHS Foundation Trust GSK VARSITY: PHD STUDENTSHIP Funder reference: 300003198

Graph to show numbers of cells captured in 3D gelatin scaffold ('Gelfoam')

<p><b>Copyright information:</b></p><p>Taken from "Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2"</p><p>http://www.biomedcentral.com/1471-2474/8/120</p><p>BMC Musculoskeletal Disorders 2007;8():120-120.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2244625.</p><p></p> It can be seen that cells were detected in the Gelfoam scaffold at days 11 and 20 in all three culture conditions. At day 11 and day 20 there were significantly reduced numbers of chondrocytes in the Gelfoam in the samples cultured in the presence of 100 ng/ml BMP-2 (*). At day 20 there was a significant decrease in cell numbers in all three experimental conditions

Graph to show numbers of cells observed on the articular cartilage surface

<p><b>Copyright information:</b></p><p>Taken from "Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2"</p><p>http://www.biomedcentral.com/1471-2474/8/120</p><p>BMC Musculoskeletal Disorders 2007;8():120-120.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2244625.</p><p></p> It can be seen that cells were observed on the articular surface at days 11 and 20 in all three culture conditions. At day 11 and day 20 there was a significant decrease in the number of cells on the cartilage surface in the samples cultured in the presence of 100 ng/ml BMP-2 (*) compared to both control and SIL sections

Graph to show numbers of cells extruding out of the articular cartilage surface

<p><b>Copyright information:</b></p><p>Taken from "Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2"</p><p>http://www.biomedcentral.com/1471-2474/8/120</p><p>BMC Musculoskeletal Disorders 2007;8():120-120.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2244625.</p><p></p> It can be seen that cells were observed to be extruding at days 11 and 20 in all three culture conditions. At day 11 there was a significant decrease in the number of cells extruding from the cartilage surface in the samples cultured in the presence of 100 ng/ml BMP-2 (*) compared to both control and SIL sections

Histological section showing the junction between the articular surface and the gelfoam scaffold

<p><b>Copyright information:</b></p><p>Taken from "Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2"</p><p>http://www.biomedcentral.com/1471-2474/8/120</p><p>BMC Musculoskeletal Disorders 2007;8():120-120.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2244625.</p><p></p> Cells have migrated out of the cartilage (bottom left of picture) and are clearly seen associated with the gelatin 'fibres' The arrow marks the Gelfoam-cartilage junction. Stained with H&E. ×200

Histological section of cartilage after SIL and culture in the presence of BMP-2 for 20 days

<p><b>Copyright information:</b></p><p>Taken from "Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2"</p><p>http://www.biomedcentral.com/1471-2474/8/120</p><p>BMC Musculoskeletal Disorders 2007;8():120-120.</p><p>Published online 5 Dec 2007</p><p>PMCID:PMC2244625.</p><p></p> A normal rounded chondrocytes is seen (black arrow) in the same field as elongated chondrocytes (red arrows). Stained with H&E. ×300