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Bone has great self-healing capacity, but above a certain critical size, bone defects will not heal spontaneously, requiring intervention to achieve full healing. Among the synthetic calcium phosphate (CaP) bone replacement materials, brushite (CaHPO4·2H2O)-based materials are of particular interest because of their degree of solubility and the related high potential to promote bone regeneration after dissolution. They can be produced tailor-made using modern three-dimensional (3D) printing technology. Although this type of implant has been widely tested in vitro, there are only limited in vivo data and less so in a relevant large animal model. In this study, material properties of a 3D-printed brushite-based scaffold are characterized, after which the material is tested by in vivo orthotopic implantation in the equine tuber coxae for 6 months. The implantation procedure was easy to perform and was well tolerated by the animals, which showed no detectable signs of discomfort. In vitro tests showed that compressive strength along the vertical axis of densely printed material was around 13 MPa, which was reduced to approximately 8 MPa in the cylindrical porous implant. In vivo, approximately 40% of the visible volume of the implants was degraded after 6 months and replaced by bone, showing the capacity to stimulate new bone formation. Histologically, ample bone ingrowth was observed. In contrast, empty defects were filled with fibrous tissue only, confirming the material’s osteoconductive capacity. It is concluded that this study provides proof that the 3D-printed brushite implants were able to promote new bone growth after 6 months’ implantation in a large animal model and that the new equine tuber coxae bone model that was used is a promising tool for bone regeneration studies. El hueso tiene una gran capacidad de autocuración, pero por encima de un cierto tamaño crítico, los defectos óseos no se curan espontáneamente, por lo que es necesario intervenir para lograr una curación completa. Entre los materiales sintéticos de sustitución ósea de fosfato de calcio (CaP), los materiales a base de brusquitos (CaHPO4-2H2O) son de particular interés por su grado de solubilidad y el elevado potencial que presentan para promover la regeneración ósea después de la disolución. Pueden producirse a medida utilizando la moderna tecnología de impresión tridimensional (3D). Aunque este tipo de implante ha sido ampliamente probado in vitro, sólo hay datos limitados in vivo y menos en un modelo relevante de animal grande. En este estudio se caracterizan las propiedades del material de un andamiaje tridimensional impreso a base de grafito, tras lo cual el material se prueba mediante la implantación ortotópica in vivo en el tubérculo equino coxae durante 6 meses. El procedimiento de implantación fue fácil de realizar y fue bien tolerado por los animales, que no mostraron ningún signo detectable de molestia. Las pruebas in vitro demostraron que la resistencia a la compresión a lo largo del eje vertical del material densamente impreso era de alrededor de 13 MPa, que se redujo a aproximadamente 8 MPa en el implante cilíndrico poroso. En vivo, aproximadamente el 40% del volumen visible de los implantes se degradó después de 6 meses y fue reemplazado por hueso, lo que demuestra la capacidad de estimular la formación de nuevo hueso. Histológicamente, se observó un amplio crecimiento del hueso. En cambio, los defectos vacíos se llenaron sólo con tejido fibroso, confirmando la capacidad osteoconductiva del material. Se llega a la conclusión de que este estudio aporta pruebas de que los implantes de grafito tridimensional fueron capaces de promover el crecimiento de nuevo hueso después de 6 meses de implantación en un modelo de animal grande y que el nuevo modelo de hueso de tubérculo equino coxae que se utilizó es una herramienta prometedora para los estudios de regeneración ósea. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Aim: The horse joint, due to its similarity with the human joint, is the ultimate model for translational articular cartilage repair studies. This study was designed to determine the critical size of cartilage defects in the equine carpus and serve as a benchmark for the evaluation of new cartilage treatment options. Material and Methods: Circular full-thickness cartilage defects with a diameter of 2, 4, and 8 mm were created in the left middle carpal joint and similar osteochondral (3.5 mm in depth) defects in the right middle carpal joint of 5 horses. Spontaneously formed repair tissue was examined macroscopically, with MR and mu CT imaging, polarized light microscopy, standard histology, and immunohistochemistry at 12 months. Results: Filling of 2 mm chondral defects was good (77.8 +/- 8.5%), but proteoglycan depletion was evident in Safranin-O staining and gadolinium-enhanced MRI (T-1Gd). Larger chondral defects showed poor filling (50.6 +/- 2.7% in 4 mm and 31.9 +/- 7.3% in 8 mm defects). Lesion filling in 2, 4, and 8 mm osteochondral defects was 82.3 +/- 3.0%, 68.0 +/- 4.6% and 70.8 +/- 15.4%, respectively. Type II collagen staining was seen in 9/15 osteochondral defects but only in 1/15 chondral defects. Subchondral bone pathologies were evident in 14/15 osteochondral samples but only in 5/15 chondral samples. Although osteochondral lesions showed better neotissue quality than chondral lesions, the overall repair was deemed unsatisfactory because of the subchondral bone pathologies. Conclusion: We recommend classifying 4 mm as critical osteochondral lesion size and 2 mm as critical chondral lesion size for cartilage repair research in the equine carpal joint model.

Bone is a dynamic tissue adapting to loading according to “Wolff's law of bone adaptation.” During very early life, however, such a mechanism may not be adequate enough to adapt to the dramatic change in environmental challenges in precocial species. Their neonates are required to stand and walk within hours after birth, in contrast to altricial animals that have much more time to adapt from the intrauterine environment to the outside world. In this study, trabecular bone parameters of the talus and sagittal ridge of the tibia from stillborn but full‐term precocials (calves and foals) were analyzed by micro‐CT imaging in order to identify possible anticipatory mechanisms to loading. Calculated average bone volume fraction in the Shetland pony (49–74%) was significantly higher compared to Warmblood foals (28–51%). Bovine trabecular bone was characterized by a low average bone volume fraction (22–28%), however, more directional anisotropy was found. It is concluded that anticipatory strategies in skeletal development exist in precocial species, which differ per species and are most likely related to anatomical differences in joint geometry and related loading patterns. The underlying regulatory mechanisms are still unknown, but they may be based on a genetic blueprint for the development of bone. More knowledge, both about a possible blueprint and its regulation, will be helpful in understanding developmental bone and joint diseases. J. Morphol. 277:948–956, 2016. © 2016 Wiley Periodicals, Inc.

Gelatine methacryloyl (GelMA) hydrogels are widely used in studies aimed at cartilage regeneration. However, the endotoxin content of commercially available GelMAs and gelatines used in these studies is often overlooked, even though endotoxins may influence several cellular functions. Moreover, regulations for clinical use of biomaterials dictate a stringent endotoxin limit. We determined the endotoxin level of five different GelMAs and evaluated the effect on the chondrogenic differentiation of equine mesenchymal stromal cells (MSCs). Cartilage-like matrix production was evaluated by biochemical assays and immunohistochemistry. Furthermore, equine peripheral blood mononuclear cells (PBMCs) were cultured on the hydrogels for 24 h, followed by the assessment of tumour necrosis factor (TNF)-&alpha, and C&ndash, C motif chemokine ligand (CCL)2 as inflammatory markers. The GelMAs were found to have widely varying endotoxin content (two with &gt, 1000 EU/mL and three with &lt, 10 EU/mL), however, this was not a critical factor determining in vitro cartilage-like matrix production of embedded MSCs. PBMCs did produce significantly higher TNF-&alpha, and CCL2 in response to the GelMA with the highest endotoxin level compared to the other GelMAs. Although limited effects on chondrogenic differentiation were found in this study, caution with the use of commercial hydrogels is warranted in the translation from in vitro to in vivo studies because of regulatory constraints and potential inflammatory effects of the content of these hydrogels.

The use of extracellular vesicles (EVs) as a potential therapy is currently explored for different disease areas. When it comes to the treatment of joint diseases this approach is still in its infancy. As in joint diseases both inflammation and the associated articular tissue destruction are important factors, both the immune-suppressive and the regenerative properties of EVs are potentially advantageous characteristics for future therapy. There is, however, only limited knowledge on the basic features, such as numerical profile and function, of EVs in joint articular tissues in general and their linking medium, the synovial fluid, in particular. Further insight is urgently needed in order to appreciate the full potential of EVs and to exploit these in EV-mediated therapies. Physiologic joint homeostasis is a prerequisite for proper functioning of joints and we postulate that EVs play a key role in the regulation of joint homeostasis and hence can have an important function in re-establishing disturbed joint homeostasis, and, in parallel, in the regeneration of articular tissues. In this mini-review EVs in the joint are explained from a historical perspective in both health and disease, including the potential niche for EVs in articular tissue regeneration. Furthermore, the translational potential of equine models for human joint biology is discussed. Finally, the use of MSC-derived EVs that is recently gaining ground is highlighted and recommendations are given for further EV research in this field.

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone.

Background In today’s porcine industry, lameness has a major welfare and economic impact, and is often caused by osteochondrosis (OC). The etiological factors of the disease have been studied in depth, however, to this day, little is known about the natural course of the disorder and how it can be detected at an early stage in pigs. The aim of this pilot study was to assess the potential of three non-invasive techniques for the detection and monitoring of early OC processes in piglets. A group of weaned piglets (n = 19) were examined longitudinally using radiographs, a visual lameness scoring scheme and a quantitative pressure-mat based locomotion analysis system to detect OC in the humeroradial, femoropatellar and tarsocrural joints. At several time points, a selection of animals was euthanized for post-mortem examinations, including histology, which was the gold standard. Results In this study, clear signs of subclinical signs of OC were observed, however, we were unsuccessful in producing clinical OC. Lesions were observed to be commonly bilaterally symmetric in the joints examined in 80 % of cases. The radiographic examinations showed a clear correlation with the gold standard, particularly when subclinical lesions were of a high histological score. Moreover, radiography was also able to detect the early repair processes, which appeared to take place at least until 14 weeks of age. Both visual scoring and pressure mat analyses showed good intra-assay reproducibility, with the pressure mat showing intra-class correlation values between 0.44 and 0.6 and the inter-observer agreement of visual scoring method was between 88 and 96 %, however their correlation to OC lesions detected by histology was very weak, with only 2 out of 12 traits for the visual scoring method showing significant and biologically logical relations to a specific joint having histological OC lesions. For the pressure mat, only a maximum of 5 associations for specific joints with histological OC lesions were found out of a possible 8. Conclusion All tested in-vivo methods showed good reproducibility. Radiography was the most reliable technique to detect and monitor longitudinally the earliest signs of OC in these piglets. It also demonstrated that the “Point of No Return” (PNR) of the disease, when repair processes end, might be later than anticipated, after 13 weeks of age. All in all, our study shows that the timing of the use of these in-vivo methods is critical to detect and monitor OC, especially in the early phases of the disease. It also shows the difficulty in producing OC regardless of the optimization of the experimental settings in relation to the etiological factors known to induce OC. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0682-z) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.