Contrast arthrography of the equine temporomandibular joint.

Background: Disorders of the equine temporomandibular joint (TMJ) cause clinical problems and detailed investigations of this joint are becoming more common. Specialist radiographic projections have the potential to highlight osseous abnormalities; however, the ability to assess the intra-articular soft tissue structures is currently limited to computed tomography (with, or without contrast enhancement) or magnetic resonance imaging. Both modalities are expensive and not readily accessible. Objective: To develop a technique of contrast arthrography of both compartments of the equine TMJ in cadavers and then perform the refined technique in three living horses as a proof-of-principle. Study design: A descriptive, experimental, study. Methods: Contrast arthrography of the discomandibular and discotemporal joint compartments of both TMJs was performed in 12 cadaveric equine heads using needles placed in the caudal pouches of the respective joint compartments. Radiographs were taken using previously published techniques, repeated with the mouth open and after air had been injected into the joints, to perform a double-contrast study. The TMJs of three healthy horses were subsequently examined to determine the validity of the procedure in live animals. Results: Single and double-contrast arthrography allowed delineation of the dorsal and ventral surfaces of the intra-articular disc in addition to filling the rostral and caudal joint pouches of the independent joint compartments. Contrast extravasation was common, and in two instances iatrogenic disc penetration resulted in the false diagnosis of pathologic disc perforation. The techniques were well tolerated in all three live horses. Main limitations: Low number of horses. Conclusion: Contrast arthrography allows interpretation of intra-articular soft tissue structures, but caution is advised in diagnosing intra-articular disc perforation. Even with experience, accessing the discomandibular joint can be challenging.

The effect of acute equine temporomandibular joint inflammation on response to rein-tension and kinematics.

Background: Although the temporomandibular joint (TMJ) is the major contact point between the reins in the riders' hand, the bit in the mouth, and the rest of the horse under saddle, the role of inflammation of this joint on equine locomotion and rein tension is unknown. Objective: To determine the effect of acute TMJ inflammation on rein-tension and horse movement when horses were long-reined on a treadmill. Study design: A randomized, controlled, cross-over design. Methods: Five horses were trained by one clinician to walk and trot on a treadmill wearing long-reining equipment instrumented with a rein-tension device and reflective optical tracking markers. Subjective assessment of horse's dominant side, and movement, were determined without rein-tension (free walk and trot); and with rein-tension (long-reined walk and trot). Continuous rein-force data from both sides were collected over ~60s from each trial. Movement was recorded using a 12-camera optical motion capture system. One randomly assigned TMJ was subsequently injected with lipopolysaccharide and the treadmill tests repeated by investigators blinded to treatment side. A second, identical assessment was performed 10 days later with the opposite TMJ being the target of intervention. Results: All horses showed reduced rein-tension on the injected (inflamed) side. Increased rein-tension was required on the non-injected side at trot, to maintain them in the correct position on the treadmill post-injection. The only kinematic variable to show any significant change due to rein tension or TMJ inflammation during the walk or trot was an increase in forward head tilt in the presence of rein tension in the trot after injection. Main limitations: Low number of horses and investigation of response to acute inflammation only. Conclusion: TMJ inflammation changed, subjectively and objectively, the response to rein-input, but the horses did not become lame.

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo.

Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is "proof of concept" that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.

Evaluation of equine synovial-derived extracellular matrix scaffolds seeded with equine synovial-derived mesenchymal stem cells.

OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 106 cells/0.5 mL) by use of a 30% serum gradient. Samples were evaluated on days 0, 3, 7, and 14. Cell migration, differentiation, and distribution into the synECMs were determined by cell surface marker CD90, viability, histologic morphology, and fluorescence microscopy results and expression of GFP, BMP-2, hyaluronan (HA), and proteoglycan (PG). RESULTS At day 14, synMSCs were viable and had multiplied 2.5-fold in the synECMs. The synECMs seeded with synMSCs had a significant decrease in CD90 expression and significant increases in HA and PG expression. The synECMs seeded with synMSCs cotransduced with GFP, or BMP-2 had a significant increase in BMP-2 expression. CONCLUSIONS AND CLINICAL RELEVANCE The synECM seeded with synMSCs or synMSCs cotransduced with GFP, or BMP-2 yielded a bioactive synovial scaffold. Expression of BMP-2 by synMSCs cotransduced to enhance expression of BMP-2 or GFP and an accompanying increase in both HA and PG expression indicated production of anabolic agents and synoviocyte differentiation in the scaffold. Because BMP-2 can promote repair of damaged cartilage, such a bioactive scaffold could be useful for treatment of injured cartilage.

Equine Dental Pulp Connective Tissue Particles Reduced Lameness in Horses in a Controlled Clinical Trial.

OBJECTIVE: To assess if injection of allogeneic dental pulp tissue particles would improve lameness in horses with naturally occurring osteoarthritis (OA) or soft tissue (ST) injury. DESIGN: Prospective, randomized, blinded, and controlled clinical trial and client survey assessment. ANIMALS: Forty lame client-owned horses. PROCEDURES: Sterile dental pulp, recovered from otherwise healthy foals that perish during dystocia, was processed under good manufacturing processing to produce mechanically manipulated, unexpanded pulp tissue particles containing viable cells surrounded in extracellular matrix. Forty lame client-owned horses with confirmed OA (n = 20), or ST injury (desmitis or tendonitis) received a 2 mL intra-articular (n = 20 OA) or intra-lesional (n = 20) injection of control transport vehicle (n = 20) or 10 × 106 dental pulp tissue particles (n = 20). Acclimatized horses had baseline measurements performed and were then injected on day 0. Horses were treadmill exercised for 2 weeks, evaluated by clinical parameters, lameness score, edema (score and circumference), pain on flexion (OA) or pressure (ST), and clients' scores for pain and discomfort before and through 45 days after pulp injection. Twenty horses were available for >2.5-year follow-up. RESULTS: Pulp-treated horses showed decrease in lameness compared to baseline (P < 0.009) or placebo controls (P < 0.013) for at least 2 weeks. Client assessments of comfort were improved between before and 45 days after pulp injection (P < 0.001). Clinical improvement with ST injury was significantly greater than OA (P < 0.001). At >2.5-year follow-up, at least 10 horses were in work. CONCLUSION AND CLINICAL RELEVANCE: Dental pulp tissue particles can be considered as a treatment option for equine lameness due to OA, desmitis, or tendonitis.

Comparison of four methods for generating decellularized equine synovial extracellular matrix.

OBJECTIVE To evaluate 4 methods for generating decellularized equine synovial extracellular matrix. SAMPLE Villous synovium harvested from the femoropatellar and medial femorotibial joints of 4 healthy adult horses < 7 years of age. Synovial samples were frozen (-80°C) until used. PROCEDURES Synovial samples were thawed and left untreated (control) or decellularized with 1 of 4 methods (15 samples/horse/method): incubation in 0.1% peracetic acid (PAA), incubation in 0.1% PAA twice, incubation in 1% Triton X-100 followed by incubation in DNase, and incubation in 2M NaCl followed by incubation in DNase. Control and decellularized samples were examined for residual cells, villous integrity, and collagen structure and integrity by means of histologic examination and scanning electron microscopy; cell viability was evaluated by means of culture and exclusion staining. Decellularization efficiency was assessed by testing for DNA content and DNA fragment size. RESULTS Incubation in PAA once preserved the synovial villous architecture, but resulted in high DNA content and retention of large (> 25,000 base pair) DNA fragments. Incubation in Triton and incubation in NaCl resulted in low DNA content and short (< 200 base pair) DNA fragments, but destroyed the synovial villous architecture. Incubation in PAA twice resulted in low DNA content and short DNA fragments while retaining the synovial villous architecture. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that of the methods evaluated, incubation in 0.1% PAA twice was the best method for generating decellularized equine synovial extracellular matrix.

Effects of repetition within trials and frequency of trial sessions on quantitative parameters of vertical force peak in horses with naturally occurring lameness.

OBJECTIVE To analyze the effects of vertical force peak (VFP) of repition within trials and between trial sessions in horses with naturally occurring appendicular lameness. ANIMALS 20 lame horses acclimated to trotting over a force plate. PROCEDURES Kinetic gait data were collected by use of a force plate regarding affected and contralateral limbs of lame horses that completed 5 valid repetitions in each of 5 sessions performed at 0, 3, 6, 12, and 24 hours, constituting 1 trial/horse. Data were compared within and among repetitions and sessions, and factors influencing VFP values were identified. RESULTS VFP values differed for lame limbs after 3 valid repetitions were performed within a session and when the interval between sessions was 3 hours. Direction of change reflected less lameness (greater VFP). Lamer horses (≥ grade 4/5) had this finding to a greater degree than did less lame horses. Results were similar for contralateral limbs regarding valid repetitions within a session; however, VFP decreased when the interval between sessions exceeded 6 hours. The coefficient of variation for VFP was ≤ 8% within sessions and ≤ 6% between sessions. The asymmetry index for VFP did not change throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE Lameness profiles obtained through kinetic gait analysis of horses with naturally occurring lameness were most accurate when valid repetitions were limited to 3 and the interval between sessions within a trial was > 3 hours. Findings suggested that natural lameness may be as suitable as experimentally induced lameness for lameness research involving horses.