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Objective Injury of articular cartilage is common, and due to the poor intrinsic capabilities of chondrocytes, it can precipitate joint degradation and osteoarthritis (OA). Implantation of autologous chondrocytes into cartilaginous defects has been used to bolster repair. Accurate assessment of the quality of repair tissue remains challenging. This study aimed to investigate the utility of noninvasive imaging modalities, including arthroscopic grading and optical coherence tomography (OCT) for assessment of early cartilage repair (8 weeks), and MRI to determine long-term healing (8 months). Design Large (15 mm diameter), full-thickness chondral defects were created on both lateral trochlear ridges of the femur in 24 horses. Defects were implanted with autologous chondrocytes transduced with rAAV5-IGF-I, autologous chondrocytes transduced with rAAV5-GFP, naïve autologous chondrocytes, or autologous fibrin. Healing was evaluated at 8 weeks post-implantation using arthroscopy and OCT, and at 8 months post-implantation using MRI, gross pathology, and histopathology. Results OCT and arthroscopic scoring of short-term repair tissue were significantly correlated. Arthroscopy was also correlated with later gross pathology and histopathology of repair tissue at 8 months post-implantation, while OCT was not correlated. MRI was not correlated with any other assessment variable. Conclusions This study indicated that arthroscopic inspection and manual probing to develop an early repair score may be a better predictor of long-term cartilage repair quality following autologous chondrocyte implantation. Furthermore, qualitative MRI may not provide additional discriminatory information when assessing mature repair tissue, at least in this equine model of cartilage repair.

The objectives of this study were to evaluate temporal changes in lubricin, hyaluronan (HA), and HA molecular weight (MW) distributions in three distinct models of equine joint injury affecting the carpal (wrist), tarsal (ankle), and femoropatellar (knee) joints. To establish ranges for lubricin, HA, and HA MW distributions across multiple joints, we first evaluated clinically healthy, high-motion equine joints. Synovial fluid was collected from high-motion joints in horses without clinical signs of joint disease (n = 11 horses, 102 joints) and from research horses undergoing carpal osteochondral fragmentation (n = 8), talar cartilage impact injury (n = 7), and femoral trochlear ridge full-thickness cartilage injury (n = 22) prior to and following arthroscopically induced joint injury. Lubricin and HA concentrations were measured via enzyme-linked immunosorbent assays, and gel electrophoresis was performed to evaluate HA MW distributions. Synovial fluid parameters were analyzed via linear regression models, revealing that lubricin and HA concentrations were conserved across healthy, high-motion joints. Lubricin concentrations increased post-injury in all osteoarthritis models (carpal fragmentation P = .001; talar impact P < .001; femoral trochlear ridge cartilage defect P = .03). Sustained loss of HA was noted post-arthroscopy following carpal osteochondral fragmentation (P < .0001) and talar impact injury (P < .001). Lubricin may be elevated to compensate for the loss of HA and to protect cartilage post-injury. Further investigation into the mechanisms regulating lubricin and HA following joint injury and their effects on joint homeostasis is warranted, including whether lubricin has value as a biomarker for post-traumatic osteoarthritis.

Temporomandibular joint (TMJ) luxation in the horse is a rare condition. There are few reports of unilateral luxation with successful reduction; bilateral luxation with concurrent bilateral coronoid fracture has not been reported. The diagnosis and closed reduction technique for rostral TMJ luxation are reported, along with the clinical outcome. A horse with an abnormally open positioned jaw presented for evaluation. Clinical exam showed a fixed open mouth, unable to be manually closed. Radiography showed multiple fractures associated with left and right TMJs. CT provided a more complete appraisal of the luxation and associated coronoid fracture orientation, as well as hyoid apparatus malposition (ie, temporohyoid subluxation). Closed reduction was achieved in this case using a fulcrum speculum between the molars, and a reduction device to forcibly appose the rostral mandible to the premaxilla with the patient anaesthetised. Functional mastication was a positive outcome; a partially phthisical, blind left eye persisted.

Objective Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long-term expression of therapeutic proteins within the joint. This study was undertaken to investigate the use of a helper-dependent adenovirus (HDAd)-mediated intraarticular gene therapy approach for long-term expression of interleukin-1 receptor antagonist (IL-1Ra) as sustained symptomatic and disease-modifying therapy for OA. Methods In mouse models of OA, efficacy of HDAd-IL-1Ra was evaluated by histologic analysis, micro-computed tomography (micro-CT), and hot plate analysis. In a horse OA model, safety and efficacy of HDAd-IL-1Ra were evaluated by blood chemistry, analyses of synovial fluid, synovial membrane, and cartilage, and gross pathology and lameness assessments. Results In skeletally immature mice, HDAd-IL-1Ra prevented development of cartilage damage, osteophytes, and synovitis. In skeletally immature and mature mice, treatment with HDAd-interleukin-1 receptor antagonist post-OA induction resulted in improved-albeit not significantly-cartilage status assessed histologically and significantly increased cartilage volume, cartilage surface, and bone surface covered by cartilage as assessed by micro-CT. Fewer osteophytes were observed in HDAd-IL-1Ra-treated skeletally immature mice. Synovitis was not affected in skeletally immature or mature mice. HDAd-IL-1Ra protected against disease-induced thermal hyperalgesia in skeletally mature mice. In the horse OA model, HDAd-IL-1Ra therapy significantly improved lameness parameters, indicating efficient symptomatic treatment. Moreover, macroscopically and histologically assessed cartilage and synovial membrane parameters were significantly improved, suggesting disease-modifying efficacy. Conclusion These data from OA models in small and large animals demonstrated safe symptomatic and disease-modifying treatment with an HDAd-expressing IL-1Ra. Furthermore, this study establishes HDAd as a vector for joint gene therapy.

BACKGROUND Following joint trauma, a posttraumatic inflammatory cascade drives degeneration of the joint. We aimed to assess whether transduction of chondrocytes with AAV5 overexpressing the immunomodulatory cytokine IL-10 would have protective effects in pellet cultures stimulated with IL-1β. METHODS Chondrocytes were isolated from 3 healthy horses and were transduced with AAV5-IL-10 at a dose of 1 x 105vg/cell. Chondrocyte pellets were formed by centrifugation and were stimulated with IL-1β starting 48 hours following transduction. After 2, 6 and 14 days in culture, supernatants were collected for cytokine analysis and RNA was isolated from cells for gene expression analysis. Pellets were also collected for biochemical analysis. RESULTS Transduction of chondrocytes led to significant increases in IL-10 expression. IL-10 expression was further enhanced by IL-1β stimulation. IL-10 overexpression led to significantly decreased expression of IL-1β and ADAMTS4. PGE2 synthesis was also significantly decreased. IL-1β mediated suppression of GAG synthesis was not rescued by IL-10. CONCLUSIONS Overexpression of IL-10 modulates the inflammatory response in chondrocytes, which may mitigate some of the deleterious effects of pro-inflammatory cascades in the posttraumatic joint. AAV5-IL-10 led to efficient and sustained overexpression of IL-10 in chondrocytes and could represent a viable treatment option for preventing osteoarthritis.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art., Involved in various diseases, hyaluronic acid is an important indicator of pathophysiology. Here, the authors report on a solid-state nanopore for the detection of the molecular weight and abundance of hyaluronic acid and demonstrate the system by studying an equine model of osteoarthritis

Summary Objective The goals of this study were (1) to quantify proteoglycan 4 ( PRG4 ) gene expression; (2) to assess lubricin immunostaining; and (3) to measure synovial fluid lubricin concentrations in clinical and experimental models of equine carpal osteoarthritis (OA). Design Lubricin synovial fluid concentrations and cartilage and synovial membrane PRG4 expression were analyzed in research horses undergoing experimental OA induction ( n = 8) and in equine clinical patients with carpal OA ( n = 58). Lubricin concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay, and PRG4 expression was quantified using qRT-PCR. Lubricin immunostaining was assessed in synovial membrane and osteochondral sections in the experimental model. Results Lubricin concentrations increased in synovial fluid following induction of OA, peaking at 21 days post-operatively in OA joints vs sham-operated controls (331 ± 69 μg/mL vs 110 ± 19 μg/mL, P = 0.001). Lubricin concentrations also increased in horses with naturally occurring OA as compared to control joints (152 ± 32 μg/mL vs 68 ± 4 μg/mL, P = 0.003). Synovial membrane PRG4 expression increased nearly 2-fold in naturally occurring OA ( P = 0.003), whereas cartilage PRG4 expression decreased 2.5-fold ( P = 0.025). Lubricin immunostaining was more pronounced in synovial membrane from OA joints as compared to controls, with intense lubricin localization to sites of cartilage damage. Conclusions Although PRG4 gene expression decreases in OA cartilage, synovial membrane PRG4 expression, synovial fluid lubricin concentrations and lubricin immunostaining all increase in an equine OA model. Lubricin may be elevated to protect joints from post-traumatic OA.

Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra-articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD-labelled MSCs [QD-MSCs]) in vivo after intra-articular injection into normal and osteoarthritic joints. One week after injection of QD-MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD-MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD-MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD-MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle-injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD-MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD-MSCs can be visualized in histological sections 1 week after intra-articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long-term tracking of MSCs, especially under proliferative conditions.

Damage to the articular cartilage surface is common in the equine athlete and, due to the poor intrinsic healing capabilities of cartilage, can lead to osteoarthritis (OA). Joint disease and OA are the leading cause of retirement in equine athletes and currently there are no effective treatments to stop the progression of OA. Several different cell-based strategies have been investigated to bolster the weak regenerative response of chondrocytes. Such techniques aim to restore the articular surface and prevent further joint degradation. Cell-based cartilage repair strategies include enhancement of endogenous repair mechanisms by recruitment of stem cells from the bone marrow following perforation of the subchondral bone plate; osteochondral implantation; implantation of chondrocytes that are maintained in defects by either a membrane cover or scaffold, and transplantation of mesenchymal stem cells into cartilage lesions. More recently, bioengineered cartilage and scaffoldless cartilage have been investigated for enhancing repair. This review article focuses on the multitude of cell-based repair techniques for cartilage repair across several species, with special attention paid to the horse.

Galectins are potent regulators of cell adhesion, growth and apoptosis in diverse cell types, including chondrocytes and synovial fibroblasts. Elevations in synovial fluid galectin-3 have been observed in rheumatoid arthritis, juvenile idiopathic arthritis and experimental inflammatory arthritis in animal models, whereas galectin-1 is thought to be protective. Less is known about galectins-1 and-3 in osteoarthritis (OA). Therefore, the purpose of this study was: (1) to determine whether galectin-1 and-3 synovial fluid concentrations and synovial membrane and cartilage histochemical staining were altered following osteochondral injury in an experimental equine osteoarthritis (OA) model and (2) to measure galectin-1 and-3 mRNA expression and synovial fluid concentrations in naturally occurring equine carpal OA. Synovial fluid galectin-1 and-3 concentrations were quantified using custom ELISAs in two research horse cohorts undergoing experimental OA induction (n = 5 and 4) and in a cohort of horses with naturally occurring carpal OA (n = 57). Galectin mRNA expression in synovial membrane and cartilage tissue obtained from carpal joints of horses with naturally occurring OA was measured using RT-qPCR, and galectin immunostaining was assessed in synovial membrane and osteochondral tissues in the experimental model (n = 5). Synovial fluid galectin-1 and-3 concentrations increased following experimental carpal osteochondral fragmentation. Cartilage galectin-1 mRNA expression increased with OA severity in naturally occurring disease. The superficial zone of healthy articular cartilage stained intensely for galectin-3 in sham-operated joints, whereas galectin-1 staining was nearly absent. Chondrocyte galectin-1 and-3 immunoreactivity increased following cartilage injury, particularly in galectin-1 positive chondrones. Galectins-1 and-3 are present in healthy equine synovial fluid and increase following post-traumatic OA. Healthy superficial zone chondrocytes express galectin-3, whereas galectin-1 chondrocyte staining is limited predominantly to chondrones and injured cartilage. Further work is needed to clarify the functions of galectins-1 and-3 in healthy and OA joints.

The objective of this study was to examine temporal variations in synovial fluid composition and lubrication following articular fracture. Post-traumatic osteoarthritis (PTOA) was induced by creating an osteochondral fracture in the middle carpal joint of four horses while the contralateral limb served as a sham-operated control. Horses were exercised on a high-speed treadmill, and synovial fluid was collected pre-operatively and at serial timepoints until 75 days post-operatively. Lubricin and hyaluronic acid (HA) concentrations were measured using sandwich ELISAs, and the molecular weight distribution of HA was analyzed via gel electrophoresis. Synovial fluid viscosity and cartilage friction coefficients across all modes of lubrication were measured on days 0, 19, 33, and 61 using a commercial rheometer and a custom tribometer, respectively. HA concentrations were significantly decreased post-operatively, and high molecular weight HA (>6.1MDa) did not recover to pre-operative values by the study termination at day 75. Lubricin concentrations increased after surgery to a greater extent in the OA as compared to sham-operated limbs. Viscosity was significantly reduced after surgery. While boundary and elastoviscous mode friction coefficients did not vary, the transition number, representing the shift between these modes, was lower. Although more pronounced in the OA limbs, similar derangements in HA, HA molecular weight distribution, viscosity, and transition number were observed in the sham-operated limbs, which may be explained by synovial fluid washout during arthroscopy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Objective To determine the prognosis for racing of horses surgically treated for slab fractures of the third carpal bone (C3). Study design Retrospective case study. Animals Horses (n = 125) surgically treated for C3 slab fractures. Methods Medical records of horses surgically treated for dorsal or sagittal C3 fractures were reviewed for age, sex, breed, limb, fracture type, degree of cartilage damage, and surgical treatment. Radiographs were evaluated to determine fracture depth, width, and displacement. Osteophytes, C3 lysis, and fragmentation were scored. Racing performance was obtained from online databases. Univariable and multivariable analyses were used to determine associations between independent variables and outcomes. Results Fifty-four (43%) horses raced postoperatively. Among thoroughbreds, 35% (30/86) with dorsal fractures and 63% (17/27) with sagittal fractures raced postoperatively. Among standardbreds, 77% (10/13) with dorsal fractures and 0% (0/2) with sagittal fractures raced postoperatively. Fracture displacement, C3 lysis, and cartilage damage affected the likelihood of racing postoperatively. Placement of 3.5-mm screws vs 4.5-mm screws and the placement of fewer screws were associated with improved likelihood of racing. Conclusion The prognosis for postoperative racing of thoroughbreds with dorsal C3 fractures was less favorable than that previously reported. Concurrent joint pathology, such as cartilage damage at time of surgery, affected the ability of the horse to race postoperatively. Clinical significance Although internal fixation of C3 slab fractures is required to restore joint congruity, return to racing should be expected in only 42% of thoroughbreds and 67% of standardbreds.

Autologous chondrocyte implantation (ACI) using a collagen scaffold (matrix-induced ACI; MACI) is a next-generation approach to traditional ACI that provides the benefit of autologous cells and guided tissue regeneration using a biocompatible collagen scaffold. The MACI implant also has inherent advantages including surgical implantation via arthroscopy or miniarthrotomy, the elimination of periosteal harvest, and the use of tissue adhesive in lieu of sutures. This study evaluated the efficacy of the MACI implant in an equine full-thickness cartilage defect model at 1 year.Autologous chondrocytes were seeded onto a collagen type-I/III membrane and implanted into one of two 15-mm defects in the femoral trochlear ridge of 24 horses. Control defects either were implanted with cell-free collagen type-I/III membrane (12 horses) or were left ungrafted as empty defects (12 horses). An additional 3 horses had both 15-mm defects remain empty as nonimplanted joints. The repair was scored by second-look arthroscopy (12 weeks), and necropsy examination (53 weeks). Healing was assessed by arthroscopic scoring, gross assessment, histology and immunohistology, cartilage matrix component assay, and gene expression determination. Toxicity was examined by prostaglandin E2 formation in joint fluid, and lymph node morphology combined with histologic screening of organs.MACI-implanted defects had improved gross healing and composite histologic scores, as well as increases in chondrocyte predominance, toluidine blue-stained matrix, and collagen type-II content compared with scaffold-only implanted or empty defects. There was minimal evidence of reaction to the implant in the synovial membrane (minor perivascular cuffing), subchondral bone, or cartilage. There were no adverse clinical effects, signs of organ toxicity, or evidence of chondrocytes or collagen type-I/III membrane in draining lymph nodes.The MACI implant appeared to improve cartilage healing in a critical-sized defect in the equine model compared with collagen matrix alone.These results indicate that the MACI implant is quick to insert, provides chondrocyte security in the defect, and improves cartilage healing compared with ACI.

There has been much interest in using autologous chondrocytes in combination with scaffold materials to aid in cartilage repair. In the present study, a total of 27 animals were used to compare the performance of matrix-assisted chondrocyte implantation (MACI®) using a collagen sponge as a chondrocyte delivery vehicle, the sponge membrane alone, and empty controls. A total of three distinct types of mechanical analyses were performed on repaired cartilage harvested from horses after 53 weeks of implantation: (1) compressive behavior of samples to measure aggregate modulus (HA) and hydraulic permeability (k) in confined compression; (2) local and global shear modulus using confocal strain mapping; and (3) boundary friction coefficient using a custom-built tribometer. Cartilage defects receiving MACI® implants had equilibrium modulus values that were 70% of normal cartilage, and were not statistically different than normal tissue. Defects filled with Maix™ membrane alone or left empty were only 46% and 51-63% of control, respectively. The shear modulus of tissue from all groups of cartilage defects were between 4 and 10 times lower than control tissue, and range from 0.2 to 0.4 MPa. The average values of boundary mode friction coefficients of control tissue from all groups ranged from 0.42 to 0.52. This study represents an extensive characterization of the mechanical performance of the MACI® grafts implant in a large animal model at 53 weeks. Collectively, these data demonstrate a range of implant performance, revealing similar compressive and frictional properties to native tissue, with inferior shear properties.

Summary Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI ® ) in critical sized defects in the equine model. Methods Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. Results MACI ® implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. Conclusions The MACI ® implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

Intraarticular (IA) administration of viral vectors expressing a therapeutic transgene is an attractive treatment modality for osteoarthritis (OA) as the joint can be treated as a contained unit. Humoral and cell-mediated immune responses in vivo can limit vector effectiveness. Transduction of articular tissues has been investigated; however, the immune response to IA vectors remains largely unknown. We hypothesized that IA rAAV2 and rAAV5 overexpressing insulin-like growth factor-I (IGF-I) would result in long-term IGF-I formation but would also induce neutralizing antibodies (NAb) and anti-capsid effector T cells. Twelve healthy horses were assigned to treatment (rAAV2 or rAAV5) or control (saline) groups. Middle carpal joints were injected with 5×10(11) vector genomes/joint. Synovial fluid was analyzed for changes in composition, NAb titers, immunoglobulin isotypes, proinflammatory cytokines, and IGF-I. Serum was analyzed for antibody titers and cytokines. A T cell restimulation assay was used to assess T cell responses. Injection of rAAV2- or rAAV5-IGF-I did not induce greater inflammation compared with saline. Synovial fluid IGF-I was significantly increased in both rAAV2- and rAAV5-IGF-I joints by day 14 and remained elevated until day 56; however, rAAV5 achieved the highest concentrations. A capsid-specific T cell response was not noted although all virus-treated horses had increased NAbs in serum and synovial fluid after treatment. Taken together, our data show that IA injection of rAAV2- or rAAV5-IGF-I does not incite a clinically detectable inflammatory or cell-mediated immune response and that IA gene therapy using minimally immunogenic vectors represents a clinically relevant tool for treating articular disorders including OA.

Background Mesenchymal stromal cells (MSCs) can be used intra-articularly to quell inflammation and promote cartilage healing; however, mechanisms by which MSCs mitigate joint disease remain poorly understood. Galectins, a family of β-galactoside binding proteins, regulate inflammation, adhesion and cell migration in diverse cell types. Galectin-1 and galectin-3 are proposed to be important intra-articular modulators of inflammation in both osteoarthritis and rheumatoid arthritis. Here, we asked whether equine bone marrow-derived MSCs (BMSCs) express higher levels of galectin-1 and -3 relative to synovial fibroblasts and chondrocytes and if an inflammatory environment affects BMSC galectin expression and motility. Methods Equine galectin-1 and -3 gene expression was quantified using qRT-PCR in cultured BMSCs, synoviocytes and articular chondrocytes, in addition to synovial membrane and articular cartilage tissues. Galectin gene expression, protein expression, and protein secretion were measured in equine BMSCs following exposure to inflammatory cytokines (IL-1β 5 and 10 ng/mL, TNF-α 25 and 50 ng/mL, or LPS 0.1, 1, 10 and 50 μg/mL). BMSC focal adhesion formation was assessed using confocal microscopy, and BMSC motility was quantified in the presence of inflammatory cytokines (IL-1β or TNF-α) and the pan-galectin inhibitor β-lactose (100 and 200 mM). Results Equine BMSCs expressed 3-fold higher galectin-1 mRNA levels as compared to cultured synovial fibroblasts (p = 0.0005) and 30-fold higher galectin-1 (p

Tendons are formed during the second half of embryonic developmentwhen tendon precursor cells deposit narrow-diameter (∼30 nm) collagenfibrils that are parallel to the long axis of the tissue. During post nataldevelopment, the narrow fibrils are replaced by large-diameter (up to500 nm)fibrils.Theabilityoftendontotransmitforcefrommuscletobone,and to dissipate forces during locomotion, is directly attributable to thecollagen fibrils. How the fibrils are synthesised, how they are alignedparallel to the tendon long axis, and how this arrangement can bereinstated during tendon healing are poorly understood. Ultrastructuralstudies of tendon lesions show the reappearance of narrow-diametercollagen fibrils and cells with slender cytoplasmic protrusions (calledfibripositors) that normally only occur in tendon during embryonicdevelopment. Recapitulation of development is a hypothesis that isgaining increasing support from researchers of tendon disease. A betterunderstanding of the genetic, molecular and environmental cues duringembryonicdevelopmentisexpectedtoprovidebetterinsightsintohowtoimprove the rate and fidelity of tendon repair in mature horses. Tendondevelopment can conveniently be considered to have an early ‘cellular’phaseandasubsequent‘matrix’phase.Inthematrix-dominatedphaseoftendon development 3D scanning electron microscopy of mouse tendonsuggests that fibripositors of the cells are the site of new fibril formationandthemechanicalinterfacebetweenthecellandtheextracellularmatrix.It is hypothesised that fibripositors exert pulling forces on collagen fibrils,and their cellular forces require functional myosin II, which is anintracellular molecular motor that is part of the actinomyosin system. Adetailed understanding of how cells set the tensional homeostasis oftendon is expected to lead to new strategies for regulating collagen fibrilassemblyinhealthandintendinopathy.

To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs).MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X).alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×10(5) cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels.equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin’s mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.

Summary Reasons for performing study: To evaluate the long-term clinical outcome after allogeneic chondrocyte and insulin-like growth factor-I (IGF-I) grafting of subchondral cystic lesions (SCLs) of the femoral condyle in horses. Objective: To test the hypothesis that chondrocyte and IGF-I grafts will improve the long-term clinical outcome in arthroscopically debrided SCLs. Methods: Medical records of 49 horses with SCLs of the femoral condyle treated by debridement and implantation of chondrocytes and IGF-I were reviewed. Preoperative radiographs were obtained, and caudocranial radiographic projections were used to establish a ratio between cyst and femoral condyle size. Arthroscopic cyst debridement followed by filling of the bone void with autologous cancellous bone (45 horses) or tricalcium phosphate granules (4 horses) was performed. A paired syringe containing a fibrinogen and chondrocyte mixture in one syringe and calcium-activated bovine thrombin with IGF-I in the other was used to cover the surface. A successful outcome was defined as a horse that performed to its intended use without lameness. Results: A successful outcome was achieved in 36 of 49 horses (74%). Preoperative radiography was performed in all horses, with 33 horses having unilateral SCLs of the medial femoral condyle, 15 horses having bilateral SCLs of the medial femoral condyle, and one horse having bilateral SCLs of the lateral femoral condyle. Median age of the horses was 3.3 years. Fifteen horses had preoperative radiographic and arthroscopic evidence of osteoarthritis (OA). A successful outcome was not influenced by age of horse, presence of pre-existing osteoarthritis or preoperative size of the subchondral cyst. Grafting resulted in success for 80% of horses >3 years old, and in 80% of horses with OA. Conclusions: Implantation of allogeneic chondrocytes supplemented with IGF-I is an effective treatment for horses with SCLs of the femoral condyle, and particularly for older horses and horses with pre-existing osteoarthritis. Potential relevance: Chondrocyte implantation may offer a greater chance of long-term success in older horses and horses with osteoarthritis than has been previously reported with cyst debridement alone.

Summary Reasons for performing study: A consistent and clinically relevant model for the induction of core lesions confined to the mid-metacarpal superficial digital flexor tendon (SDFT) has not been previously reported. Injection of bacterial collagenase is commonly used but often results in large, irregular and inconsistent lesions that disrupt the superficial tendon layers and epitenon. Objective: To develop and evaluate a new injection technique for collagenase induction of SDFT injury. Methods: Collagenase gel was injected into a physical columnar defect created by longitudinally placing a curved 16 gauge 8.89 cm needle in the mid-metacarpal SDFT in a randomly selected forelimb of 10 horses. A placebo treatment injection was performed 1 week later. Serial ultrasound examinations were performed. Horses were subjected to euthanasia at 2 (n = 2), 4 (n = 2), 8 (n = 4) and 16 (n = 2) weeks post treatment injection. Post mortem magnetic resonance imaging and histological analysis were performed. Gene expression (18S, SCX, TNC, TNMD, COL1A1, COL3A1, COMP, DCN, MMP1, MMP3 and MMP13), total DNA, glycosaminoglycan and collagen content were determined for experimental tendons (n = 10) and unaffected tendons (n = 9). Results: Mid-metacarpal SDFT core lesion induction was successful in all tendons with consistent lesion cross-sectional area and minimal epitenon disruption. Histology confirmed loss of normal tendon architecture after tendonitis induction and subsequent healing of the tendon core lesion. Compared with gene expression in unaffected tendons, several tested genes were significantly upregulated (COL1A1, COL3A1, TNMD, SCX, TNC, MMP13), while others showed significant downregulation (COMP, DCN, and MMP3). Conclusion: Compared with the previously used direct injection of collagenase, this injection technique was easily performed and induced more consistent lesions that were mid-metacarpal and did not disrupt the epitenon. Potential relevance: This model will allow for objective assessment of therapies for tendon regeneration in the mid-metacarpal SDFT prior to clinical trials and routine clinical application.

Summary Reasons for performing study: Long-term efficacy of arthroscopic cartilage reattachment for the treatment of osteochondritis dissecans (OCD) lesions in the equine femoropatellar joint is unknown. Objective: To evaluate radiographic outcome and long-term performance of horses undergoing OCD reattachment. Hypothesis: Separated OCD cartilage flaps may be reincorporated into the joint surface by reattachment rather than flap removal. Methods: Polydioxanone pins were utilised arthroscopically to reattach OCD lesions in 40 of 44 joints from 27 horses. Cartilage was reattached when it had persisting perimeter continuity, the surface was not deeply fissured or irregular, and the cartilage was not protuberant or extensively mineralised. Bone marrow aspirate concentrate was grafted to additional areas denuded of cartilage or alongside reattached cartilage. Results: Breeds included Thoroughbred (n = 18), Quarter Horse (n = 4), Warmblood (n = 3), Standardbred (n = 1) and Arabian (n = 1). Mean age was 9.7 months. Radiographic lesion length was 1.5–6.3 cm. Reattachment alone was used in 32 of 44 affected joints, a combination of debridement and reattachment in 8 joints and debridement alone in 4 joints. One horse was destroyed due to tendon laceration. Of the remaining 26 horses, mean duration of follow-up was 15.6 months (range 2 months–12 years). Radiographic resolution of OCD lesions treated with reattachment was significantly improved at 6 months. Twenty horses had long-term performance data, of which 19 were sound and had reached intended athletic potential. One horse remained lame, and an additional 6 were sound but remained unbroken or were convalescing. Thus, an overall success rate based upon continued soundness in performing horses was 95% (19/20). Conclusion: Cartilage flap reattachment can salvage OCD cartilage by integration with the underlying bone. Potential relevance: Extensive OCD cartilage flaps may be salvaged by reattachment which can result in normal radiographic subchondral bone contour and long-term athletic performance.

Objective: To determine in vitro elution characteristics of amikacin and ticarcillin from fiber reinforced calcium phosphate beads (FRCP). Sample Population: Experimental. Methods: FRCP beads with water (A), amikacin (B), ticarcillin/clavulanate (C), or both amikacin and ticarcillin/clavulanate (D) were bathed in mL phosphate-buffered saline (PBS) at 37°C, 5% CO2 and 95% room air. PBS was sampled (eluent) and beads were placed in fresh PBS at time points 1 and 8 hours and 1, 2, 3, 4, 5, 6, 7, 10, 12, 14, 18, 21, 25, 28, 35, 42, 49, and 56 days. Antibiotic concentration and antimicrobial activity of eluent against Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were determined. Results: Both antibiotics eluted in a bimodal pattern. Beads with a single antibiotic eluted 20.8 ± 2.5% of amikacin and 29.5 ± 0.8% of ticarcillin over 56 days. Coelution of the antibiotics resulted in a lower proportion (AUC0–∞) of antibiotics eluted for both amikacin (9.5 ± 0.2%) and ticarcillin (21.7 ± 0.09%). Bioassay of antimicrobial activity of the eluent (t=1, 8, and 24 hours) established reduced antimicrobial activity of amikacin from combination beads (D). Conclusions: FRCP beads with amikacin or ticarcillin/clavulanate, but not the combination, are suitable carriers for wound implantation. Clinical Relevance: Duration before complete resorption of FRCP beads in vivo should be determined before clinical use as a resorbable depot. The results of this study underscore the importance of testing drug combinations, despite success of the combination systemically, before their use in local applications.

Objective—To investigate the efficacy and safety of a low-volume, single-catheter, continuous peripheral neural blockade (CPNB) technique to locally deliver bupivacaine to alleviate signs of severe forelimb pain resulting from experimentally induced tendonitis in horses. Design—Randomized controlled experimental trial. Sample—14 horses and 5 forelimbs from equine cadavers. Procedures—Horses underwent collagenase-induced superficial digital flexor tendonitis in the midmetacarpal region of 1 forelimb. To deliver analgesia, a closed-tip catheter was placed from lateral to medial, approximately 12 cm distal to the accessory carpal bone, between the suspensory ligament and accessory ligament of the deep digital flexor tendon. Success of catheter placement and anesthetic delivery was documented ex vivo in 5 forelimbs from equine cadavers. Effective analgesia in affected forelimbs of horses from continuous (n = 7) versus intermittent (7) local anesthetic delivery (intermittent peripheral neural blockade; IPNB) was compared over a 3-day period. Results—Horses that received CPNB in the affected forelimb were less lame than horses that received IPNB. A lower proportion of CPNB-treated horses had behavioral and physiologic signs of pain, compared with IPNB-treated horses. Neither technique completely blocked the sensation of pain or resulted in swelling in the distal portion of the forelimb, vasodilation, or an increase in lameness. After removal, Staphylococcus aureus was cultured from 1 catheter tip. Conclusions and Clinical Relevance—For short-term treatment, CPNB was more effective than IPNB for reduction in signs of severe pain in the distal aspect of the forelimb of horses.

Case Description—3 horses were referred for treatment of subchondral cystic lesions of 1 or both medial femoral condyles. Clinical Findings—All horses had clinically apparent lameness confirmed to be due to a radiographically evident subchondral cystic lesion of the medial femoral condyle with a large articular component (> 15 mm) and shallow subchondral depth (< 10 mm). Arthroscopic assessment of affected cartilage revealed undulating cartilage with a relatively smooth surface and extensive residual perimeter attachment. Treatment and Outcome—Resorbable polydioxanone pins were used arthroscopically to reattach the cartilage overlying the subchondral cystic lesions. A biologic graft (bone marrow aspirate concentrate or allogeneic chondrocytes) was injected into the depths of the cystic cavity following cartilage reattachment. Follow-up examination confirmed radiographic resolution of the lesion and elimination of clinical signs within the treated femorotibial joint. Clinical Relevance—Lesions with a large area of affected articular cartilage have been associated with a decreased rate of return to athletic function following arthroscopic debridement, likely secondary to the loss of subchondral architecture and the production of imperfect fibrocartilage repair. Salvage of the affected cartilage in a select population of horses with progressively expanding but shallow subchondral cystic lesions of the medial femoral condyle is possible and may improve radiographic and clinical outcome.