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The objective of this study was to evaluate a non-resorbable implant for the focal repair of chondral defects in eight adult horses with 12-month follow-up. The bi-layered construct composed of a polycarbonate-urethane-urea biomaterial which was printed in 3D fashion onto a bone anchor was implanted into surgically created osteochondral defects into the femoropatellar joints of eight horses. The analysis of post-mortem outcomes were compared to defects treated with microfracture in the same animal on the contralateral femoropatellar jointfemoropatellar joint. The overall macroscopic scoring after 12 months yielded higher scores in the OCI-treated stifles compared to MF treatment (p = 0.09) with better quality and filling of the defect. Histology revealed good anchorage of repair tissue growing into the 3D structure of the implant and histopathology scoring for adjacent native cartilage showed no difference between groups. MRI and micro-CT showed overall less sclerotic reactions in the surrounding bone in the implant group and no foreign body reaction was detected. Biomechanical analysis of the repair tissue revealed a significantly higher peak modulus (p < 0.05) in the implant group (0.74 ± 0.45) compared to the microfracture control group (0.15 ± 0.11). Dynamic loading yielded higher values for the repair tissue overgrowing the implant group (0.23 ± 0.17) compared to the microfracture control (0.06 ± 0.06) (p < 0.05). The bi-layered osteochondral implant provided a safe implant for focal repair of full-thickness osteochondral defects, as no adverse reaction was seen within the joints and the level of degeneration of adjacent cartilage to the repair site was not different compared to that seen in defects treated with microfracture after 12 months., (© 2024 The Author(s). Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Strategies for articular cartilage repair need to take into account topographical differences in tissue composition and architecture to achieve durable functional outcome. These have not yet been investigated in the equine stifle., Objectives: To analyse the biochemical composition and architecture of three differently loaded areas of the equine stifle. We hypothesise that site differences correlate with the biomechanical characteristics of the cartilage., Study Design: Ex vivo study., Methods: Thirty osteochondral plugs per location were harvested from the lateral trochlear ridge (LTR), the distal intertrochlear groove (DITG) and the medial femoral condyle (MFC). These underwent biochemical, biomechanical and structural analysis. A linear mixed model with location as a fixed factor and horse as a random factor was applied, followed by pair-wise comparisons of estimated means with false discovery rate correction, to test for differences between locations. Correlations between biochemical and biomechanical parameters were tested using Spearman's correlation coefficient., Results: Glycosaminoglycan content was different between all sites (estimated mean [95% confidence interval (CI)] for LTR 75.4 [64.5, 88.2], for intercondylar notch (ICN) 37.3 [31.9, 43.6], for MFC 93.7 [80.1109.6] μg/mg dry weight), as were equilibrium modulus (LTR2.20 [1.96, 2.46], ICN0.48 [0.37, 0.6], MFC1.36 [1.17, 1.56] MPa), dynamic modulus (LTR7.33 [6.54, 8.17], ICN4.38 [3.77, 5.03], MFC5.62 [4.93, 6.36] MPa) and viscosity (LTR7.49 [6.76, 8.26], ICN16.99 [15.88, 18.14], MFC8.7 [7.91,9.5]°). The two weightbearing areas (LTR and MCF) and the non-weightbearing area (ICN) differed in collagen content (LTR 139 [127, 152], ICN176[162, 191], MFC 127[115, 139] μg/mg dry weight), parallelism index and angle of collagen fibres. The strongest correlations were between proteoglycan content and equilibrium modulus (r: 0.642; p: 0.001), dynamic modulus (r: 0.554; p < 0.001) and phase shift (r: -0.675; p < 0.001), and between collagen orientation angle and equilibrium modulus (r: -0.612; p < 0.001), dynamic modulus (r: -0.424; p < 0.001) and phase shift (r: 0.609; p < 0.001)., Main Limitations: Only a single sample per location was analysed., Conclusions: There were significant differences in cartilage biochemical composition, biomechanics and architecture between the three differently loaded sites. The biochemical and structural composition correlated with the mechanical characteristics. These differences need to be acknowledged by designing cartilage repair strategies., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

In this study, we mapped and quantified changes of proteoglycan (PG) content and biomechanical properties in articular cartilage in which either blunt or sharp grooves had been made, both close to the groove and more remote of it, and at the opposing joint surface (kissing site) in equine carpal joints. In nine adult Shetland ponies, standardized blunt and sharp grooves were surgically made in the radiocarpal and middle carpal joints of a randomly chosen front limb. The contralateral control limb was sham-operated. At 39 weeks after surgery, ponies were euthanized. In 10 regions of interest (ROIs) (six remote from the grooves and four directly around the grooves), PG content as a function of tissue-depth and distance-to-groove was estimated using digital densitometry. Biomechanical properties of the cartilage were evaluated in the six ROIs remote from the grooves. Compared to control joints, whole tissue depth PG loss was found in sites adjacent to sharp and, to a larger extent, blunt grooves. Also, superficial PG loss of the surgically untouched kissing cartilage layers was observed. Significant PG loss was observed up to 300 µm (sharp) and at 500 µm (blunt) from the groove into the surrounding tissue. Equilibrium modulus was lower in grooved cartilage than in controls. Grooves, in particular blunt grooves, gave rise to severe PG loss close to the grooved sites and to mild degeneration more remote from the grooves in both sharply and bluntly grooved cartilage and at the kissing sites, resulting in loss of mechanical strength over the 9-month period., (© 2022. The Author(s).)

Background: High-power laser therapy gained popularity recently as a regenerative treatment for tendinitis and desmitis in the horse. However, studies evaluating the effects of laser therapy on tissue repair at the histological level in large mammals are lacking., Objectives: To evaluate the effects of high-power laser therapy on suspensory desmitis healing, using a model of suspensory ligament branch injury., Study Design: In vivo experiments., Methods: Standardised lesions were surgically induced in all four lateral suspensory branches of 12 healthy Warmblood horses. Laser therapy (class 4, 15W) was applied daily on two of four induced lesions for four consecutive weeks. Horses were randomly assigned to either short-term study (horses were sacrificed after 4 weeks) or long-term study (6 months). Suspensory ligament samples were scored after staining with haematoxylin-eosin and immunostaining for collagen 1- collagen 3- and factor VIII., Results: In the short-term study, significantly better (lower) scores for variation in density (17% above cut-off score in treated lesions vs. 31% above cut-off score in controls, P = .03), shape of nuclei (54% vs 92%, P = .02), fibre alignment (32% vs 75%, P = .003) and fibre structure (38% vs 71%, P = .02) were found in laser-treated lesions when compared to controls. Collagen 3 expression was significantly higher (32% vs 19%, P = .006) in control lesions. In both short- and long-term studies combined, parameters lesion size (44% vs 56%, P = .02) and shape of nuclei (53% vs 84%, P = .05) scored significantly better in treated lesions. Long-term, significantly better (lower) scores were found in the laser-treated group for lesion size (15% vs 45%, P = .008) and a higher percentage above cut-off score for density of the nuclei (27% vs 9%, P = .02), compared to controls., Main Limitations: The model of suspensory branch injury is not an exact representation of clinical overstrain lesions., Conclusions: These results suggest that high-power laser therapy enables better lesion healing than conservative treatment., (© 2022 EVJ Ltd.)

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and euthanized at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contra-lateral sham-operated controls (p < 0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a threefold to sevenfold change in interleukin-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid extracted from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited., (© 2020 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

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 (CaHPO 4 ·2H 2 O)-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.

Cartilage repair remains a major challenge and treatment of (osteo)chondral defects generally results in poor quality fibrous repair tissue. Our approach aims to address some of the major biomechanical issues encountered in scaffold-based cartilage repair, such as insufficient stiffness of the scaffolds, step formation at the interface with the native tissue and inadequate integration with the original tissue. Two osteochondral defects were created on the medial femoral trochlear ridge in each stifle of six Shetland ponies. The defects were filled with a bi-layered implant consisting of a polyetherketoneketone (PEKK) bone anchor and a polyurethane elastomer. The defects in the contralateral joint served as unfilled controls. After 12 weeks, the ponies were euthanased and tissues were evaluated macroscopically and using micro-computed tomography, histology and immunohistochemistry. Post-operative recovery was good in all ponies and minimal lameness was observed. After 12 weeks, the proximally located plug was partially covered (mean±standard deviation [SD] percentage surface area covered 72.5±19.7%) and the distal plug was nearly completely covered (mean±SD percentage surface area covered 98.5±6.1%) with stiff and smooth repair tissue. Histology and immunohistochemistry confirmed that the repair tissue was well connected to the native cartilage but contained negligible amounts of collagen type II and glycosaminoglycans (GAGs). The repair tissue was stiff and fibrous in nature and presented a nearly flush surface with the surrounding native cartilage distally. This approach therefore resolves a number of issues related to scaffold-based cartilage repair and compares favourably with results of several other studies in large animal models. However, long-term follow-up is needed to evaluate the true potential of this type of implant., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Extracellular vesicle (EV) concentration, characteristics and function in equine synovial fluid (SF) during normal growth and development has not previously been studied. Isolation of EVs was performed in SF from three healthy foals and two adult horses by differential ultracentrifugation (10,000g and 200,000g); EVs were purified by sucrose density gradient floatation and analysed by high-resolution flow cytometry (FCM), buoyant density and western blotting. Additionally, repeated biomarker analysis of sulphated glycosaminoglycans (GAG), matrix metalloproteinase (MMP), C-terminal crosslinked telopeptide type II collagen (CTX-II), collagenase cleaved neopeptide type II collagen (C2C) was performed in SF from 10 foals and six adult horses. In contrast with the quantitative EV profile, the biomarker profile in SF from juvenile joints was substantially different from that in SF from adult animals. However, there were qualitative differences in the high-resolution FCM scatter plots. Future in-depth functional analyses may reveal differences between juvenile and mature EVs in SF., (Copyright © 2018. Published by Elsevier Ltd.)

Controlled biomaterial-based corticosteroid release might circumvent multiple injections and the accompanying risks, such as hormone imbalance and muscle weakness, in osteoarthritic (OA) patients. For this purpose, microspheres were prepared from an amino acid-based polyester amide (PEA) platform and loaded with triamcinolone acetonide (TAA). TAA loaded microspheres were shown to release TAA for over 60days in PBS. Furthermore, the bioactivity lasted at least 28days, demonstrated by a 80-95% inhibition of PGE 2 production using TNFα-stimulated chondrocyte culture, indicating inhibition of inflammation. Microspheres loaded with the near infrared marker NIR780-iodide injected in healthy rat joints or joints with mild collagenase-induced OA showed retention of the microspheres up till 70days after injection. After intra-articular injection of TAA-loaded microspheres, TAA was detectable in the serum until day seven. Synovial inflammation was significantly lower in OA joints injected with TAA-loaded microspheres based on histological Krenn scores. Injection of TAA-loaded nor empty microspheres had no effect on cartilage integrity as determined by Mankin scoring. In conclusion, the PEA platform shows safety and efficacy upon intra-articular injection, and its extended degradation and release profiles compared to the currently used PLGA platforms may render it a good alternative. Even though further in vivo studies may need to address dosing and readout parameters such as pain, no effect on cartilage pathology was found and inflammation was effectively lowered in OA joints., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To investigate the effect of decellularized cartilage-derived matrix (CDM) scaffolds, by itself and as a composite scaffold with a calcium phosphate (CaP) base, for the repair of osteochondral defects. It was hypothesized that the chondral defects would heal with fibrocartilaginous tissue and that the composite scaffold would result in better bone formation., Methods: After an 8-week pilot experiment in a single horse, scaffolds were implanted in eight healthy horses in osteochondral defects on the medial trochlear ridge of the femur. In one joint a composite CDM-CaP scaffold was implanted (+P), in the contralateral joint a CDM only (-P) scaffold. After euthanasia at 6 months, tissues were analysed by histology, immunohistochemistry, micro-CT, biochemistry and biomechanical evaluation., Results: The 8-week pilot showed encouraging formation of bone and cartilage, but incomplete defect filling. At 6 months, micro-CT and histology showed much more limited filling of the defect, but the CaP component of the +P scaffolds was well integrated with the surrounding bone. The repair tissue was fibrotic with high collagen type I and low type II content and with no differences between the groups. There were also no biochemical differences between the groups and repair tissue was much less stiff than normal tissue (P < 0.0001)., Conclusions: The implants failed to produce reasonable repair tissue in this osteochondral defect model, although the CaP base in the -P group integrated well with the recipient bone. The study stresses the importance of long-term in vivo studies to assess the efficacy of cartilage repair techniques., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The presence of Chlamydia pneumoniae in the human arterial system has mainly been determined in atherosclerotic plaque, whereas the adventitia has remained relatively unexplored. We assessed the presence of C. pneumoniae in all three vessel wall layers of coronary (n=72) and brachial (n=48) arteries in relation to local atherosclerosis. Immunohistochemical staining of C. pneumoniae was observed in plaque and adventitia. Cells stained for C. pneumoniae were detected in the same areas as cells stained for macrophages in adjacent sections. C. pneumoniae staining in the adventitia was associated with the extent and severity of atherosclerosis. Coronary sections with C. pneumoniae staining in both adventitia and plaque more often contained advanced atherosclerosis than sections with staining only in the adventitia. Staining was observed more often in the coronary artery than in the brachial artery (24/72 vs. 5/48 and 51/72 vs. 8/48 for plaque and adventitia, respectively, P=0.004 and P<0.001). PCR confirmed the presence of C. pneumoniae DNA in the adventitia. In summary, the adventitia of atherosclerotic coronary arteries frequently contains C. pneumoniae that seems to be located within macrophages. These results might indicate a possible route for infected circulating macrophages to home into atherosclerotic lesions in the artery via vasa vasorum.

-Retrospectively, plaque rupture is often colocalized with inflammation of the cap and shoulder of the atherosclerotic plaque. Local inflammation is therefore considered a potential marker for plaque vulnerability. However, high specificity of inflammation for plaque rupture is a requisite for application of inflammation markers to detect rupture-prone lesions. The objective of the present study was to investigate the prevalence and distribution (local versus general) of inflammatory cells in nonruptured atherosclerotic plaques. The cap and shoulder of the plaque were stained for the presence of macrophages and T lymphocytes in 282 and 262 cross sections obtained from 74 coronary and 50 femoral arteries, respectively. From most cases, 2 atherosclerotic arteries were studied to gain insight into the local and systemic distribution of the inflammatory process. In 45% and 41% of all cross sections, staining for macrophages was observed in the femoral and coronary arteries, respectively. Rupture of the fibrous cap was observed in 2 femoral and 3 coronary artery segments and was always colocalized with inflammatory cells. At least 1 cross section stained positively for CD68 or acid phosphatase in 84% and 71% of all femoral and coronary arteries, respectively. Only 1 femoral and 6 coronary arteries revealed a positive stain for CD68 in all investigated segments. Inflammation of the cap and shoulder of the plaque is a common feature, locally observed, in atherosclerotic femoral and coronary arteries. The high prevalence of local inflammatory responses should be considered if they are used as a diagnostic target to detect vulnerable, rupture-prone lesions.

T lymphocytes were propagated in vitro from endomyocardial biopsy specimens that were obtained weekly from four patients during the first 2 to 3 months after heart transplantation. The culture was performed in the presence of recombinant interleukin-2 and interleukin-4, with or without mitogen, in which especially CD8+ donor-specific cytotoxic T cells expanded. These cells, presumably reflecting an in vivo activated population, could even be cultured from biopsy specimens without histopathologic signs of rejection. A preferential expression of T cell receptor V beta gene families was found in these T-cell lines. This finding is in contrast with the heterogenous expression in peripheral blood T cells of the same patient. The restriction in V beta gene family expression was substantiated in the evaluation of clones obtained from two cell lines. Among 17 donor-specific cytotoxic T-cell clones derived from one cell line, only four V beta gene families were expressed. All five clones from the other cell line used the V beta 8 family. Some clones expressing a distinct V beta gene family used the same V-D-J junction sequence, indicative of their origin from the same precursor. With the use of oligonucleotide probes complementary to clone-specific V-D-J junction sequences, four of five clones were detected not only in the parent T-cell line but also in T-cell lines derived from biopsy specimens with rejection reactions taken 1 week earlier and 2 weeks later and in blood cells taken before and 0.5, 3, and 6 months after transplantation; these clones were not detected in blood cells harvested 12 months after transplantation. This study showed a restricted usage of the V beta gene families by activated donor-specific cytotoxic T lymphocytes in the heart transplant.

The cytoimmunologic monitoring assay has been proposed as a useful noninvasive technique in the diagnosis of rejection and infection after heart transplantation. In this study, we have analyzed the diagnostic usefulness of cytoimmunologic monitoring in 73 patients after heart transplantation. For individual patients, the follow-up varied between 2 and 78 months. Data were related to histopathologic characteristics of the endomyocardial biopsy. Significantly different cytoimmunologic monitoring results were not observed between groups according to endomyocardial biopsy histopathologic evaluation. The diagnostic usefulness of cytoimmunologic monitoring depended on the cutoff value applied. With higher cutoff values, the sensitivity decreased and the specificity and predictive value increased. For the previously reported cutoff value of 5%, the sensitivity was 0.29, the specificity was 0.73, and the predictive value was 0.66. Values of sensitivity, specificity, and predictive value were similar when only the first acute rejection was taken into account, or when only data on the first 4 weeks and the first 6 months after transplantation were considered. In calculating the diagnostic usefulness of the sensitivity, specificity, and predictive values were observed. We concluded that cytoimmunologic monitoring has a limited value for diagnosing acute rejection after heart transplantation.

Serial endomyocardial biopsies from 5 patients during the first 3 months after heart transplantation were studied by immunohistochemistry for the neural markers neurofilament 200 kD, neuron-specific protein 9.5 (PGP9.5), S100 (Schwann cell marker), and tyrosine hydroxylase (TH). In normal endomyocardium, nerves immunoreactive for neurofilament 200 kD and PGP9.5 occurred in the interstitium around blood vessels, in close contact with myocyte fibrils. Immunoreactive fibers identified for S100 and TH were also present. In biopsies taken after transplantation, the basic nerve structure in neurofilament labeling was intact. There was a disappearance of immunolabeling for PGP9.5, S100, and TH during the first month after transplantation. Immunoreactivity reappeared during the second month, at first in the interstitium around blood vessels. This was observed for PGP9.5 and TH between 4 and 6 weeks after transplantation, and for S100 (in two of five patients) starting after 6 weeks. There was no apparent relation between reappearance and occurrence of rejection.