Proteome and phospholipidome interrelationship of synovial fluid-derived extracellular vesicles in equine osteoarthritis: An exploratory 'multi-omics' study to identify composite biomarkers.

Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease.

Skin grafting with the modified Meek technique in the standing horse using full thickness skin: Evaluation of acceptance, wound contraction and wound closure in chronic wounds.

BACKGROUND: The modified Meek technique is not commonly used in equine wound management, despite the consistent reliable and superior results compared with other grafting techniques. Major drawbacks are the need for specialised, expensive equipment and general anaesthesia. OBJECTIVES: To describe adjustments of the modified Meek technique enabling use in the standing horse without the need for the full equipment. This implied the use of full-thickness skin grafts manually harvested from the pectoral area and manually cut into micrografts. Graft acceptance; healing progress; and final functional and cosmetic result were outcome parameters. STUDY DESIGN: Descriptive case series. METHODS: Eight horses with traumatic wounds at the dorsal side of the carpus or tarsus, healing by second intention, were treated. Original wound areas and areas of graft acceptance and rejection were determined from post-processing of digital photographs and percentage acceptance, wound contraction and epithelialisation were calculated. RESULTS: The initial mean wound area was 55.4 cm2 . Graft acceptance was 95.3 ± 2.5%. Wound closure was due to 46.0 ± 25.6% wound contraction and 54.0 ± 25.6% epithelialisation and resulted in 96.8 ± 1.9% reduction of the initial wound area 28.0 ± 8.5 days after grafting. The scar was flat, flexible and functional, usually with thin and regular hair growth. The adapted procedure was fast and efficient, with a learning curve for the increased manual work. MAIN LIMITATIONS: Small study population. CONCLUSIONS: This adapted modified Meek technique can successfully be performed in the standing horse and obviates the need for the full expensive equipment and general anaesthesia. The acceptance of the full-thickness grafts is excellent resulting in fast and satisfactory healing.

Orthotopic equine study confirms the pivotal importance of structural reinforcement over the pre-culture of cartilage implants.

In articular cartilage (AC), the collagen arcades provide the tissue with its extraordinary mechanical properties. As these structures cannot be restored once damaged, functional restoration of AC defects remains a major challenge. We report that the use of a converged bioprinted, osteochondral implant, based on a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electrowritten polycaprolactone micrometer-scale fibers in a zonal fashion, inspired by native collagen architecture, can provide long-term mechanically stable neo-tissue in an orthotopic large animal model. The design of this novel implant was achieved via state-of-the-art converging of extrusion-based ceramic printing, melt electrowriting, and extrusion-based bioprinting. Interestingly, the cell-free implants, used as a control in this study, showed abundant cell ingrowth and similar favorable results as the cell-containing implants. Our findings underscore the hypothesis that mechanical stability is more determining for the successful survival of the implant than the presence of cells and pre-cultured extracellular matrix. This observation is of great translational importance and highlights the aptness of advanced 3D (bio)fabrication technologies for functional tissue restoration in the harsh articular joint mechanical environment.

Osseous pathologic changes in the lumbar region of the equine vertebral column: A descriptive post-mortem study in three breeds.

BACKGROUND: Data on equine lumbar pathology hardly exist in breeds other than Thoroughbreds. OBJECTIVES: To describe pathological changes of the osseous lumbar vertebral column in Warmblood horses, Shetland ponies and Konik horses. STUDY DESIGN: Descriptive post-mortem study. METHODS: The lumbar vertebral columns of 34 Warmblood horses, 28 Shetland ponies, and 18 Konik horses were examined by computed tomography (CT). Osteoarthritis (OA) of articular processes (APJs), OA of intertransverse joints (ITJs), intervertebral disc (IVD) mineralisation, impingement of spinous (SPs) and transverse (TPs) processes and spondylosis were scored. Breed differences in prevalence and severity of pathologies were analysed by linear regression analysis and by calculating Spearman's rank correlation coefficients (rs ). RESULTS: In Warmblood horses, the prevalence of OA of APJs, impingement of SPs and TPs was respectively 90%, 36%, and 35%, significantly higher than in the other breeds (p < 0.001). In Konik horses, IVD mineralisation (40%) and spondylosis (10%) were more frequent than in Warmbloods and Shetland ponies (p = 0.03). Severity score for OA of ITJs was highest in Shetland ponies (p < 0.001). For impingement of SPs, severity score was highest in Warmbloods (p = 0.03), and of TPs lowest in Shetland ponies (p = 0.003). For all parameters, except for spondylosis in Shetland ponies, there was a positive correlation between percentage of vertebrae affected and age, with IVD mineralisation scores increasing faster in Konik horses (p < 0.001). In all breeds, there was also a positive relation between scores of severity and age for OA of APJs and ITJs and for IVD mineralisation, with severity scores increasing faster in Shetland ponies (p = 0.04). Strong left/right correlations of the severity scores were seen for OA of the APJ, ITJ, impingement of TPs, and paramedian spondylosis (rs = 0.74-0.86, all p < 0.001). MAIN LIMITATIONS: Clinical histories were not available. CONCLUSIONS: There are distinct breed differences between prevalence and severity of osseous pathologies of the lumbar spine. Warmblood horses have higher scores for most pathologies with IVD mineralisation being more important in Konik horses and OA of ITJs in Shetland ponies.

Microstructural differences in the osteochondral unit of terrestrial and aquatic mammals.

During evolution, animals have returned from land to water, adapting with morphological modifications to life in an aquatic environment. We compared the osteochondral units of the humeral head of marine and terrestrial mammals across species spanning a wide range of body weights, focusing on microstructural organization and biomechanical performance. Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of the microarchitecture-function relationship is pivotal for understanding articular biology and, hence, for the development of durable functional regenerative approaches for treatment of joint damage, which are thus far lacking.

A Translational Model for Repeated Episodes of Joint Inflammation: Welfare, Clinical and Synovial Fluid Biomarker Assessment.

This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.

Acute joint inflammation induces a sharp increase in the number of synovial fluid EVs and modifies their phospholipid profile.

Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo.

BACKGROUND: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. OBJECTIVES: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. STUDY DESIGN: Pre-clinical experimental study. METHODS: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = -1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. RESULTS: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. MAIN LIMITATIONS: Ex vivo study protocol and a low number of investigated joints. CONCLUSIONS: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.

Site- and Zone-Dependent Changes in Proteoglycan Content and Biomechanical Properties of Bluntly and Sharply Grooved Equine Articular Cartilage.

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.

Folate Receptor Expression by Human Monocyte-Derived Macrophage Subtypes and Effects of Corticosteroids.

OBJECTIVE: Folate receptor beta (FR-ß) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-ß+ macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-ß expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid. DESIGN: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-ß expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: FR-ß expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-ß expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-ß+ phenotype. CONCLUSIONS: As corticosteroids skewed monocytes toward an FR-ß-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-ß has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-ß expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-ß+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.

Continuous versus discrete data analysis for gait evaluation of horses with induced bilateral hindlimb lameness.

BACKGROUND: Gait kinematics measured during equine gait analysis are typically evaluated by analysing (asymmetry-based) discrete variables (eg, peak values) obtained from continuous kinematic signals (eg, timeseries of datapoints). However, when used for the assessment of complex cases of lameness, such as bilateral lameness, discrete variable analysis might overlook relevant functional adaptations. OBJECTIVES: The overall aim of this paper is to compare continuous and discrete data analysis techniques to evaluate kinematic gait adaptations to lameness. STUDY DESIGN: Method comparison. METHODS: Sixteen healthy Shetland ponies, enrolled in a research programme in which osteochondral defects were created on the medial trochlear ridges of both femurs, were used in this study. Kinematic data were collected at trot on a treadmill before and at 3 and 6 months after surgical intervention. Statistical parametric mapping and linear mixed models were used to compare kinematic variables between and within timepoints. RESULTS: Both continuous and discrete data analyses identified changes in pelvis and forelimb kinematics. Discrete data analyses showed significant changes in hindlimb and back kinematics, where such differences were not found to be significant by continuous data analysis. In contrast, continuous data analysis provided additional information on the timing and duration of the differences found. MAIN LIMITATIONS: A limited number of ponies were included. CONCLUSIONS: The use of continuous data provides additional information regarding gait adaptations to bilateral lameness that is complementary to the analysis of discrete variables. The main advantage lies in the additional information regarding time dependence and duration of adaptations, which offers the opportunity to identify functional adaptations during all phases of the stride cycle, not just the events related to peak values.

Dual-contrast computed tomography enables detection of equine posttraumatic osteoarthritis in vitro.

To prevent the progression of posttraumatic osteoarthritis, assessment of cartilage composition is critical for effective treatment planning. Posttraumatic changes include proteoglycan (PG) loss and elevated water content. Quantitative dual-energy computed tomography (QDECT) provides a means to diagnose these changes. Here, we determine the potential of QDECT to evaluate tissue quality surrounding cartilage lesions in an equine model, hypothesizing that QDECT allows detection of posttraumatic degeneration by providing quantitative information on PG and water contents based on the partitions of cationic and nonionic agents in a contrast mixture. Posttraumatic osteoarthritic samples were obtained from a cartilage repair study in which full-thickness chondral defects were created surgically in both stifles of seven Shetland ponies. Control samples were collected from three nonoperated ponies. The experimental (n = 14) and control samples (n = 6) were immersed in the contrast agent mixture and the distributions of the agents were determined at various diffusion time points. As a reference, equilibrium moduli, dynamic moduli, and PG content were measured. Significant differences (p < 0.05) in partitions between the experimental and control samples were demonstrated with cationic contrast agent at 30 min, 60 min, and 20 h, and with non-ionic agent at 60 and 120 min. Significant Spearman's rank correlations were obtained at 20 and 24 h (ρ = 0.482-0.693) between the partition of cationic contrast agent, cartilage biomechanical properties, and PG content. QDECT enables evaluation of posttraumatic changes surrounding a lesion and quantification of PG content, thus advancing the diagnostics of the extent and severity of cartilage injuries.

Sustained Intra-Articular Release and Biocompatibility of Tacrolimus (FK506) Loaded Monospheres Composed of [PDLA-PEG1000]-b-[PLLA] Multi-Block Copolymers in Healthy Horse Joints.

There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG1000]-b-PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.

Adaptation strategies of horses with induced forelimb lameness walking on a treadmill.

BACKGROUND: There is a paucity of research describing the gait pattern of lame horses at the walk. OBJECTIVES: To describe the changes in motion pattern and vertical ground reaction forces (GRFz) in horses with induced forelimb lameness at the walk and compare those changes with the changes observed at the trot. STUDY DESIGN: Experimental study. METHODS: In 10 clinically sound Warmblood horses, moderate forelimb lameness was induced using a sole pressure model followed by trot and walk on a treadmill. Kinematic data were collected using 3D optical motion capture (OMC), and GRFz by an instrumented treadmill. Mixed models were used to compare sound baseline versus forelimb lameness (significance was set at P < .05). RESULTS: Lameness induction significantly reduced peak GRFz on the second force peak, and vertical impulse in the lame limb. Stride and stance duration in all limbs were reduced. Lameness significantly affected the vertical movement symmetry of the head and withers. Maximum limb retraction angle, fetlock extension and protraction speed were reduced in the lame limb. Body centre of mass (COM) translation was reduced in the side-to-side direction and increased in the vertical and fore-aft directions. Several compensatory kinetic and kinematic changes were observed in the nonlame limbs. The observed changes in both kinetics and kinematics were generally smaller at walk with fewer variables being affected, compared to the trot. MAIN LIMITATIONS: Only one degree and type of orthopaedic pain (sole pressure) was studied. CONCLUSIONS: Compensatory strategies of forelimb lameness at the walk include alteration of several kinetic and kinematic parameters and have some specific patterns and inter-individual differences that are not seen at the trot. However, much like at the trot, head movement and forelimb vertical force symmetry seem to be the most useful parameters to detect forelimb lameness at walk.

Structural, compositional, and functional effects of blunt and sharp cartilage damage on the joint: A 9-month equine groove model study.

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.

Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces.

Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.

Long-Term in Vivo Performance of Low-Temperature 3D-Printed Bioceramics in an Equine Model.

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.

Arthroscopic Determination of Cartilage Proteoglycan Content and Collagen Network Structure with Near-Infrared Spectroscopy.

Conventional arthroscopic evaluation of articular cartilage is subjective and insufficient for assessing early compositional and structural changes during the progression of post-traumatic osteoarthritis. Therefore, in this study, arthroscopic near-infrared (NIR) spectroscopy is introduced, for the first time, for in vivo evaluation of articular cartilage thickness, proteoglycan (PG) content, and collagen orientation angle. NIR spectra were acquired in vivo and in vitro from equine cartilage adjacent to experimental cartilage repair sites. As reference, digital densitometry and polarized light microscopy were used to evaluate superficial and full-thickness PG content and collagen orientation angle. To relate NIR spectra and cartilage properties, ensemble neural networks, each with two different architectures, were trained and evaluated by using Spearman's correlation analysis (ρ). The ensemble networks enabled accurate predictions for full-thickness reference properties (PG content: ρin vitro, Val= 0.691, ρin vivo= 0.676; collagen orientation angle: ρin vitro, Val= 0.626, ρin vivo= 0.574) from NIR spectral data. In addition, the networks enabled reliable prediction of PG content in superficial (25%) cartilage (ρin vitro, Val= 0.650, ρin vivo= 0.613) and cartilage thickness (ρin vitro, Val= 0.797, ρin vivo= 0.596). To conclude, NIR spectroscopy could enhance the detection of initial cartilage degeneration and thus enable demarcation of the boundary between healthy and compromised cartilage tissue during arthroscopic surgery.

Critical-sized cartilage defects in the equine carpus.

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 µCT 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 (T1Gd). 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.