Exploring stable-based behaviour and behaviour switching for the detection of bilateral pain in equines

Efficient and sensitive animal pain detection approaches are increasingly studied with the goal of improving animal welfare and monitoring the efficacy of treatment and rehabilitation. The aim of this study was to determine the potential of various behaviours as sensitive indicators of subtle inflammation states in equines. The long-term goal of this research is to understand how to objectively and remotely classify behaviours that are associated with inflammation using wearable inertial sensor technologies. This study represents a proof-of concept investigation to ascertain what behavioural indices might be important in long-term monitoring of mild bilateral inflammation and recovery with a view to translating the approach to a technology-enabled remote monitoring paradigm. Bilateral synovitis of the intercarpal joints was induced in seven equines using lipopoly saccharide (0.25 ng) at time zero. The horses were confined to stables and monitored intermittently over seven days by stable-fixed video cameras. White blood cell count, carpal circumference and food availability were recorded across the study. An ethogram was created to manually annotate behaviours from video footage following lameness induction at seven different timepoints across a 1-week period. Behaviour data were processed extracting the duration, frequency and variability of behaviours. One-way repeated measures ANOVA revealed a significant time effect for white blood cell count and behaviour switching. There were no significant changes in carpal circumferences and heart rate measures over the sampling period. Food availability appears to be an important contextual factor that should be considered in pain-related behavioural studies. We conclude that behaviour variability may be a promising indicator of subtle bilateral inflammation which should be further explored in larger controlled trials and different pain presentations. Future work will seek to optimise grouping of behaviours associated with inflammation that can be detected using wearable technologies for future remote monitoring protocols

Chondrogenically Primed Human Mesenchymal Stem Cells Persist and Undergo Early Stages of Endochondral Ossification in an Immunocompetent Xenogeneic Model

Tissue engineering approaches using progenitor cells such as mesenchymal stromal cells (MSCs) represent a promising strategy to regenerate bone. Previous work has demonstrated the potential of chondrogenically primed human MSCs to recapitulate the process of endochondral ossification and form mature bone in vivo, using immunodeficient xenogeneic models. To further the translation of such MSC-based approaches, additional investigation is required to understand the impact of interactions between human MSC constructs and host immune cells upon the success of MSC-mediated bone formation. Although human MSCs are considered hypoimmunogenic, the potential of chondrogenically primed human MSCs to induce immunogenic responses in vivo, as well as the efficacy of MSC-mediated ectopic bone formation in the presence of fully competent immune system, requires further elucidation. Therefore, the aim of this study was to investigate the capacity of chondrogenically primed human MSC constructs to persist and undergo the process of endochondral ossification in an immune competent xenogeneic model. Chondrogenically differentiated human MSC pellets were subcutaneously implanted to wild-type BALB/c mice and retrieved at 2 and 12 weeks post-implantation. The percentages of CD4(+) and CD8(+) T cells, B cells, and classical/non-classical monocyte subsets were not altered in the peripheral blood of mice that received chondrogenic MSC constructs compared to sham-operated controls at 2 weeks post-surgery. However, MSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. Flow cytometric analysis of retrieved MSC constructs identified the presence of T cells and macrophages at 2 and 12 weeks post-implantation, with low levels of immune cell infiltration to implanted MSC constructs detected by CD45 and CD3 immunohistochemical staining. Despite the presence of immune cells in the tissue, MSC constructs persisted in vivo and were not degraded/resorbed. Furthermore, constructs became mineralised, with longitudinal micro-computed tomography imaging revealing an increase in mineralised tissue volume from 4 weeks post-implantation until the experimental endpoint at 12 weeks. These findings indicate that chondrogenically differentiated human MSC pellets can persist and undergo early stages of endochondral ossification following subcutaneous implantation in an immunocompetent xenogeneic model. This scaffold-free model may be further extrapolated to provide mechanistic insight to osteoimmunological processes regulating bone regeneration and homeostasis

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100–150 μm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.</p

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100–150 μm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.</p

Long-Term Expansion, Enhanced Chondrogenic Potential, and Suppression of Endochondral Ossification of Adult Human MSCs via WNT Signaling Modulation

Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair

An integral biochemical analysis of the main constituents of articular cartilage, subchondral and trabecular bone

AbstractObjectiveIn articular joints, the forces generated by locomotion are absorbed by the whole of cartilage, subchondral bone and underlying trabecular bone. The objective of this study is to test the hypothesis that regional differences in joint loading are related to clear and interrelated differences in the composition of the extracellular matrix (ECM) of all three weight-bearing constituents.MethodCartilage, subchondral- and trabecular bone samples from two differently loaded sites (site 1, dorsal joint margin; site 2, central area) of the proximal articular surface of 30 macroscopically normal equine first phalanxes were collected. Collagen content, cross-linking (pentosidine, hydroxylysylpyridinoline (HP), lysylpyridinoline (LP)) hydroxylation, and denaturation, as well as glycosaminoglycan (GAG) and DNA content were measured in all three tissues. In addition, bone mineral density (BMD), the percentage of ash and the mineral composition (calcium, magnesium and phosphorus) were determined in the bony samples.ResultsFor pentosidine cross-links there was an expected correlation with age. Denatured collagen content was significantly higher in cartilage at site 1 than at site 2 and was higher in trabecular bone compared to subchondral bone, with no site differences. There were significant site differences in hydroxylysine (Hyl) concentration and HP cross-links in cartilage that were paralleled in one or both of the bony layers. In subchondral bone there was a positive correlation between total (HP+LP) cross-links and Ca content. For Ca and other minerals there were corresponding site differences in both bony layers.ConclusionsIt is concluded that there are distinct differences in distribution of the major biochemical components over both sites in all three layers. These differences show similar patterns in cartilage, subchondral bone and trabecular bone, stressing the functional unity of these tissues. Overall, differences could be interpreted as adaptations to a considerably higher cumulative loading over time at site 2, requiring stiffer tissue. Turnover is higher in trabecular bone than in subchondral bone. In cartilage, the dorsal site 1 appears to suffer more tissue damage

Allogeneic Chondrogenic Mesenchymal Stromal Cells Alter Helper T Cell Subsets in CD4+ Memory T Cells

Implantation of chondrogenically differentiated mesenchymal stromal cells (MSCs) leads to bone formation in vivo through the process of endochondral ossification. The use of allogeneic MSCs for this purpose may be a promising new approach to replace the current gold standard of bone regeneration. However, the success of using allogeneic cells depends on the interaction between the implanted cells and the host's endogenous immune cells. Th17 T cells and other CD4 helper T cell subtypes have been shown to negatively impact chondrogenesis, however, it is unclear how the interaction between these cells affects bone regeneration mediated by these cells. The aim of the current work was to assess the effect of chondrogenic MSC pellets on Th1, Th2, Th17, and regulatory T cells in vitro. Human MSCs were nonchondrogenic (-TGFβ3) and chondrogenically (+TGFβ3) differentiated for 7 or 21 days. Memory T cells (sorted from the CD4 population of peripheral blood mononuclear cells [PBMCs]), as well as total PBMCs were cocultured with allogeneic nonchondrogenic and chondrogenic MSC pellets for 3 days. Seven-day differentiated allogeneic nonchondrogenic and chondrogenic MSC pellets that were cocultured with memory T cells resulted in a significant increase in Th2 and a decrease in Th1 T cells. Furthermore, the co-culture of 21-day differentiated nonchondrogenic and chondrogenic MSC pellets with memory T cells resulted in a significant increase in Th2 and Th17 T cells, as well as a decrease in Th1 and regulatory T cells. Interleukin (IL)-6 was identified as a predominant cytokine involved in this interaction between allogeneic chondrogenically differentiated MSC pellets and memory CD4 T cells, with high levels of IL-6 being secreted in the supernatants of this cocultured condition. The findings of this study highlight the potential of chondrogenically differentiated MSC pellets to alter the ratio of Th1 and Th2 as well as Th17 and regulatory T cell subsets. Additional analysis investigating bone formation by chondrogenically differentiated MSCs in an allogeneic setting may identify a novel role of these T cell subsets in bone regeneration processes mediated by chondrogenically differentiated MSCs. Allogeneic mesenchymal stromal cells (MSCs) have the potential to be an off-the-shelf treatment for bone repair. However, the lack of knowledge of the immune cells involved in this process has hampered the progression to the clinic. The current study has shown that allogeneic chondrogenic MSCs have the potential to skew the ratio of specific helper CD4 T cell subsets in vitro. This has now provided insight for future in vivo experiments to investigate the role of these T cell subsets in the early stages of bone regeneration mediated by allogeneic chondrogenic MSCs