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The aim of this study is to assess whether articular cartilage changes in an equine model of post‐traumatic osteoarthritis (PTOA), induced by surgical creation of standard (blunt) grooves, and very subtle sharp grooves, could be detected with ex vivo T1 relaxation time mapping utilizing three‐dimensional (3D) readout sequence with zero echo time. Grooves were made on the articular surfaces of the middle carpal and radiocarpal joints of nine mature Shetland ponies and osteochondral samples were harvested at 39 weeks after being euthanized under respective ethical permissions. T1 relaxation times of the samples (n = 8 + 8 for experimental and n = 12 for contralateral controls) were measured with a variable flip angle 3D multiband‐sweep imaging with Fourier transform sequence. Equilibrium and instantaneous Young's moduli and proteoglycan (PG) content from OD of Safranin‐O‐stained histological sections were measured and utilized as reference parameters for the T1 relaxation times. T1 relaxation time was significantly (p < 0.05) increased in both groove areas, particularly in the blunt grooves, compared with control samples, with the largest changes observed in the superficial half of the cartilage. T1 relaxation times correlated weakly (Rs ≈ 0.33) with equilibrium modulus and PG content (Rs ≈ 0.21). T1 relaxation time in the superficial articular cartilage is sensitive to changes induced by the blunt grooves but not to the much subtler sharp grooves, at the 39‐week timepoint post‐injury. These findings support that T1 relaxation time has potential in detection of mild PTOA, albeit the most subtle changes could not be detected. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is increasingly thought to be a multifactorial disease in which sustained gut inflammation serves as a continued source of inflammatory mediators driving degenerative processes at distant sites such as joints. The objective of this study was to use the equine model of naturally occurring obesity associated OA to compare the fecal microbiome in OA and health and correlate those findings to differential gene expression synovial fluid (SF) cells, circulating leukocytes and cytokine levels (plasma, SF) towards improved understanding of the interplay between microbiome and immune transcriptome in OA pathophysiology., Methods: Feces, peripheral blood mononuclear cells (PBMCs), and SF cells were isolated from healthy skeletally mature horses (n=12; 6 males, 6 females) and those with OA (n=6, 2 females, 4 males). Horses were determined to have OA via lameness evaluation, response to intra-articular (IA) diagnostic analgesia, and radiographic and arthroscopic evidence. Horses were excluded who had received medications or joint injections within 2 months. Cytokine analyses of plasma and SF were performed via multiplex immunoassay. Fecal bacterial microbial 16s DNA sequencing was performed and correlated to bulk RNA sequencing of SF cells and PBMC performed using an Illumina based platform., Results: Horses with OA had higher body condition scores (P=0.009). Cytokines were elevated in plasma [interleukin (IL)-2, IL-6, IL-18, interferon gamma (IFN-γ), interferon gamma inducible protein 10 (CXCL10 or IP-10), granulocyte colony-stimulating factor (G-CSF)] and SF (IL-1β, IL-6, IL-17A, IL-18, IP-10, G-CSF) in OA. Microbial principal coordinate analysis (PCoA) using Bray-Curtis dissimilarity for β-diversity demonstrated distinct grouping of samples from OA versus healthy horses (P=0.003). Faith alpha diversity was reduced in OA (P=0.02). Analysis of microbiome composition showed differential relative abundance of taxa on multiple levels in OA. Specific phyla ( Firmicutes, Verrucomicrobia, Tenericutes, Fibrobacteres ), correlated to transcriptomic differences related to cell structure, extracellular matrix, collagen, laminin, migration, and motility, or immune response to inflammation in OA., Conclusions: These findings provide compelling evidence for a link between obesity, gut microbiome dysbiosis and differential gene expression in distant joint sites associated with development of OA in a relevant large animal model, establishing a connection here that provides a platform from which development of therapeutic interventions targeting the gut microbiome can build., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-24-109/coif). L.M.P. reports that this study was funded by the Animal Health and Disease Grant (No. NI22AHDRXXXXG011) Project Accession No. 7003828 from the USDA National Institute of Food and Agriculture. Additional grant and contract funding sources include Grayson Jockey Club Research Foundation, NIH/NCATS CCTSI and the CSU Research Council. Current or pending patents unrelated to this work include CSU-SURF 2022-041, CSU-SURF 2021-053, CSU SURF 2023-025, and CSU STRATA 2024-016. The other authors have no conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Osteoarthritis (OA) is a degenerative and heterogeneous disease that affects all types of joint structures. Current clinical treatments are only symptomatic and do not manage the degenerative process in animals or humans. One of the new orthobiological treatment strategies being developed to treat OA is the use of drug delivery systems (DDS) to release bioactive molecules over a long period of time directly into the joint to limit inflammation, control pain, and reduce cartilage degradation. Two vasoactive peptides, endothelin-1 and bradykinin, play important roles in OA pathogenesis. In this study, we investigated the effects of two functionalized nanogels as DDS. We assessed the effect of chitosan functionalized with a type A endothelin receptor antagonist (BQ-123-CHI) and/or hyaluronic acid functionalized with a type B1 bradykinin receptor antagonist (R-954-HA). The biocompatibility of these nanogels, alone or in combination, was first validated on equine articular chondrocytes cultured under different oxic conditions. Further, in an OA equine organoid model via induction with interleukin-1 beta (IL-1β), a combination of BQ-123-CHI and R-954-HA (BR5) triggered the greatest decrease in inflammatory and catabolic markers. In basal and OA conditions, BQ-123-CHI alone or in equimolar combinations with R-954-HA had weak pro-anabolic effects on collagens synthesis. These new nanogels, as part of a composite DDS, show promising attributes for treating OA. [ABSTRACT FROM AUTHOR]

BackgroundAllogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation.ObjectivesTo investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation.Study DesignRandomized positively and negatively controlled experimental study.MethodIn phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology.ResultsIn phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs.Main LimitationsThis study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control.ConclusionsIn this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment.

Background: Osteoarthritis (OA) is a major source of pain and disability worldwide. Understanding of disease progression is evolving, but OA is increasingly thought to be a multifactorial disease in which the innate immune system plays a role in regulating and perpetuating low-grade inflammation. The aim of this study was to enhance our understanding of OA immunopathogenesis through characterization of the transcriptomic responses in OA joints, with the goal to facilitate the development of targeted therapies., Methods: Single-cell RNA sequencing (scRNA-seq) was completed on cells isolated from the synovial fluid of three normal and three OA equine joints. In addition to synovial fluid, scRNA-seq was also performed on synovium from one normal joint and one OA joint., Results: Characterization of 28,639 cells isolated from normal and OA-affected equine synovial fluid revealed the composition to be entirely immune cells (CD45 + ) with 8 major populations and 26 subpopulations identified. In synovial fluid, we found myeloid cells (macrophage and dendritic cells) to be overrepresented and T cells (CD4 and CD8) to be underrepresented in OA relative to normal joints. Through subcluster and differential abundance analysis of T cells we further identified a relative overrepresentation of IL23R + gamma-delta (γδ) T cells in OA-affected joints (a cell type we report to be enriched in gene signatures associated with T helper 17 mediated immunity). Analysis of an additional 17,690 cells (11 distinct cell type clusters) obtained from synovium of one horse led to the identification of an OA-associated reduction in the relative abundance of synovial macrophages, which contrasts with the increased relative abundance of macrophages in synovial fluid. Completion of cell-cell interaction analysis implicated myeloid cells in disease progression, suggesting that the myeloid-myeloid interactions were increased in OA-affected joints., Conclusions: Overall, this work provides key insights into the composition of equine synovial fluid and synovium in health and OA. The data generated in this study provides equine-specific cell type gene signatures which can be applied to future investigations. Furthermore, our analysis highlights the potential role of macrophages and IL23R + γδ T cells in OA immunopathogenesis., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-24-40/coif). Z.J.W. reports that stipend funding was provided by NIH/NCATS Colorado CTSA T32TR004366. L.M.P. reports that this study was funded by the Foundation for the Horse, Grayson Jockey Club Research Foundation, Colorado State University College Research Council Interdisciplinary Pilot Award, NIH/NCATS CTSA 5TL1TR002533-02 (L.M.P.), NIH 5T32OD010437-19 (L.M.P.), and Carolyn Quan and Porter Bennett. The other authors have no conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Objective: To determine the symptomatic and disease-modifying capabilities of sEH and COX inhibitors during joint inflammation.Methods: Using a blinded, randomized, crossover experimental design, 6 adult healthy horses were injected with lipopolysaccharide (LPS; 3 μg) from E. coli in a radiocarpal joint and concurrently received the non-selective cyclooxygenase (COX) inhibitor phenylbutazone (2 mg/kg), the sEH inhibitor t-TUCB (1 mg/kg) or both (2 mg/kg phenylbutazone and 0.1, 0.3, and 1 mg/kg t-TUCB) intravenously. There were at least 30 days washout between treatments. Joint pain (assessed via inertial sensors and peak vertical forces), synovial fluid concentrations of prostanoids (PGE2, TxB2), cytokines (IL-1β, IL-6, TNF-α) and biomarkers of collagen synthesis (CPII) and degradation (C2C) were measured at pre-determined intervals over a 48-h period. The anti-apoptotic effect of COX and sEH inhibitors was determined via ELISA technique in primary equine chondrocytes incubated with TNF-α (10 ng/ml) for 24 h. Apoptosis was also determined in chondrocytes incubated with sEH-generated metabolites.Results: Combined COX and sEH inhibition produced significantly better control of joint pain, prostanoid responses, and collagen synthesis-degradation balance compared to each compound separately. When administered separately, pain control was superior with COX vs. sEH inhibition. Cytokine responses were not different during COX and/or sEH inhibition. In cultured chondrocytes, sEH inhibition alone or combined with COX inhibition, but not COX inhibition alone had significant anti-apoptotic effects. However, sEH-generated metabolites caused concentration-dependent apoptosis.Conclusions: Combined COX and sEH inhibition optimize pain control, attenuate loss of articular cartilage matrix during joint inflammation and cytokine-induced chondrocyte apoptosis.

Infectious arthritis is difficult to treat in both human and veterinary clinical practice. Recent literature reports Staphylococcus aureus as well as other gram‐positive and gram‐negative isolates forming free‐floating biofilms in both human and equine synovial fluid that are tolerant to traditional antimicrobial therapy. Using an in vitro equine model, we investigated the ability of platelet‐rich plasma (PRP) formulations to combat synovial fluid biofilm aggregates. Synovial fluid was infected, and biofilm aggregates allowed to form over a 2‐hour period. PRP was collected and processed into different formulations by platelet concentration, leukocyte presence, and activation or lysis. Infected synovial fluid was treated with different PRP formulations with or without aminoglycoside cotreatment. Bacterial load (colony‐forming unit/mL) was determined by serial dilutions and plate counting at 8 hours posttreatment. All PRP formulations displayed antimicrobial properties; however, formulations containing higher concentrations of platelets without leukocytes had increased antimicrobial activity. Lysis of PRP and pooling of the PRP lysate (PRP‐L) from multiple horses as compared to individual horses further increased antimicrobial activity. This activity was lost with the removal of the plasma component or inhibition of the proteolytic activity within the plasma. Fractionation of pooled PRP‐L identified the bioactive components to be cationic and low‐molecular weight (<10 kDa). Overall, PRP‐L exhibited synergism with amikacin against aminoglycoside tolerant biofilm aggregates with greater activity against gram‐positive bacteria. In conclusion, the use of PRP‐L has the potential to augment current antimicrobial treatment regimens which could lead to a decrease in morbidity and mortality associated with infectious arthritis. [ABSTRACT FROM AUTHOR]

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The status of articular cartilage often defines the level, progression, and subsequent prognosis of joint disease in both human and equine athletes. Although methods to diagnose equine cartilage defects have significantly improved over the past decade, articular cartilage damage and ensuing osteoarthritis remain a challenge to treat. The following 2 categories of surgical options for cartilage repair in the horse are typically considered: palliative and reparative or restorative. Palliative surgical care consists of arthroscopic debridement and lavage, whereas reparative options involve the use of marrow stimulation techniques. Restorative or reparative surgical options are an area of active research, including the use of osteochondral grafting, autologous chondrocyte implantation (ACI), and augmentation with mesenchymal stem cells (MSCs). Subchondral bone microfracture coupled with intra-articular stem cell injection is currently considered to be the optimal treatment combination for equine patients with articular cartilage defects. Following arthroscopic surgery, the rehabilitative goals are to provide support to the affected limb, restore joint flexibility, stability, and manage perioperative pain. In addition, biologic therapy for the treatment of equine joint disease continues to be clinically employed and of investigational interest. With the large amount of ongoing in vitro research in bioprinted osteochondral constructs and potential extracellular matrix components, and the advantages of the in vivo equine model of articular cartilage repair as well as naturally occurring disease, it seems inevitable that the horse would be used to develop these novel techniques. [ABSTRACT FROM AUTHOR]

In order to determine that the stiffness varies between different joint types, an indentation test was performed on fresh equine articular cartilage surfaces from the fetlock, carpal, and stifle joints. The results demonstrated that the stiffness varies on different joints showing different mechanical behaviors. A flat-ended cylindrical indenter is lowered at a constant rate for 20 s until the indentation depth reaches 0.2 mm (velocity of 10 pm/s). It was shown that the articular cartilage of the fetlock is stiff er than the carpal and stifle joints. The average stiffness of the fetlock, carpal, and stifle joints are 46.1 Nimm, 20.5 Nimm, and 2.73 Nimm, respectively. The coefficients of a fit for the joints were statistically compared as well using the student t-test. The differences of some coefficients between the fetlock, carpus, and stifle were "very highly significant" (p <0.001). Four different surfaces in the fetlock and four in carpal joint were compared as well. The front lateral, front medial, rear lateral, and rear medial cartilage surfaces in the fetlock were not significantly different in stiffness. In the carpus, the distal radius and proximal radial carpal bone articular cartilage surfaces showed significantly different stiffness from the others, while the distal radial carpal bone and proximal third carpal bone articular cartilage swfaces possessed similar stiffness values. The cartilage surfaces from the radiocarpal joint were stiffer than the midcarpal joint. Clear trends in the correlations between stiffness and weight as well as stiffness and age of the horse were not observed. [ABSTRACT FROM AUTHOR]

Purpose: Thicknesses of fresh equine articular cartilage surfaces from the fetlock, carpal and stifle joints were measured employing a needle probe test. Materials and methods: Eighty-seven samples used in measurement were cultivated from fetlock, carpal and stifle joints of 12 deceased within 4 h of death. After approximately three minutes of exposure to air during dissection, all cartilage samples were preserved in a saline solution to keep the articular cartilage hydrated for testing. The thickness was measured on five different spots on the same sample. The thicknesses of the fetlock, carpus and stifle were compared. Results: The articular cartilage of the stifle was thicker than the fetlock and carpus, while the fetlock and the carpus had similar thickness values. The average thickness of the fetlock, carpal and stifle joint are 0.86, 0.87 and 2.1 mm, respectively. They were statistically compared using the Student t-test. The differences on the articular cartilage thicknesses between the fetlock and stifle, and carpus and stifle were 'very highly significant' ( p < 0.001). This indicates that the articular cartilage thickness of the stifle is significantly different from that of the fetlock and carpus. Four different surfaces in the fetlock and four in the carpal joint were also compared. Significant differences between each set of the four surfaces were not observed. In the carpus, the difference in thickness between the distal radius and proximal third carpal bone articular cartilage surfaces as well as the proximal radial carpal bone and distal radial carpal bone articular cartilage surfaces were statistically significant. [ABSTRACT FROM AUTHOR]

Abstract: Nutrition and management modeling has been expanding rapidly in the last 30 years. Nutrition and physiology models in ruminants have used different modeling techniques ranging from simple empirical-based to mechanistic and dynamic models. The level of complexity is driven by available data and modeler objective. The two primary objectives are to serve as a research tool to help guide future research versus field-applied models. Field-applied models combine empirical and mechanistic techniques, allowing nutritionists and consultants to evaluate and predict animal performance by using inputs that can be collected on-farm and by using standard laboratory methodology. More complex dynamic/mechanistic models have been historically used to guide research. These models have tended to focus on enhancing the biochemical understanding associated with lactating cows. Regardless of methodology, modeling has improved the nutrition and feeding practices of all species in which it has been used. However, limited modeling has occurred regarding the equine. The objectives of this article are to introduce the basis for an equine model (Fancy) and outline how modeling can be used to enhance knowledge by guiding research projects. During the development of Fancy, inconsistencies related to requirement predictions were discovered. Most inconsistencies were related to data limitations; however, this illustrates where modeling can offer significant rewards. The development of a model can assist in defining these inconsistencies and data limitations. Research resources can then be better allocated to address these issues, resulting in expanding the knowledge base of the system being modeled. [Copyright &y& Elsevier]

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1β. In some instances, alone or in the presence of IL-1β, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression. [ABSTRACT FROM AUTHOR]

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.

Objective: To report on the experiences with the use of commercial and autologous fibrin glue (AFG) and of an alternative method based on a 3D-printed polycaprolactone (PCL) anchor for the fixation of hydrogel-based scaffolds in an equine model for cartilage repair., Methods: In a first study, three different hydrogel-based materials were orthotopically implanted in nine horses for 1-4 weeks in 6 mm diameter full-thickness cartilage defects in the medial femoral trochlear ridge and fixated with commercially available fibrin glue (CFG). One defect was filled with CFG only as a control. In a second study, CFG and AFG were compared in an ectopic equine model. The third study compared the efficacy of AFG and a 3D-printed PCL-based osteal anchor for fixation of PCL-reinforced hydrogels in three horses for 2 weeks, with a 4-week follow-up to evaluate integration of bone with the PCL anchor. Short-term scaffold integration and cell infiltration were evaluated by microcomputed tomography and histology as outcome parameters., Results: The first study showed signs of subchondral bone resorption in all defects, including the controls filled with CFG only, with significant infiltration of neutrophils. Ectopically, CFG induced clear inflammation with strong neutrophil accumulation; AFG was less reactive, showing fibroblast infiltration only. In the third study the fixation potential for PCL-reinforced hydrogels of AFG was inferior to the PCL anchor. PCL reinforcement had disappeared from two defects and showed signs of dislodging in the remaining four. All six constructs fixated with the PCL anchor were still in place after 2 weeks. At 4 weeks, the PCL anchor showed good integration and signs of new bone formation., Conclusions: The use of AFG should be preferred to xenogeneic products in the horse, but AFG is subject to individual variations and laborious to make. The PCL anchor provides the best fixation; however, this technique involves the whole osteochondral unit, which entails a different conceptual approach to cartilage repair.