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 × 10 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.

Summary A horse was presented with a history of unpredictable behaviour under saddle to the point that the owner deemed the horse dangerous to ride. In addition, at rest the horse frequently protruded his tongue and during mastication a “clunking” sound could be heard. Radiographic examination at a previous clinic had revealed a normal left temporomandibular joint (TMJ) and a radiopaque mass in the right TMJ. However, despite arthroscopic surgery of the abnormal joint, the clinical signs had not resolved. Diagnostic investigation at the referral clinic revealed that TMJ analgaesia altered both a baseline hindlimb lameness and a behavioural component to the movement of the horse. Computed tomography (CT) illustrated the presence of bone cysts within the mandibular condyles and marginal osteophytosis of the discomandibular joints consistent with osteoarthritis. Arthroscopic examination, debridement of the affected joints and post‐operative intra‐articular injection of autologous protein solution resulted in temporary resolution of both the behavioural changes and the “clunking” sound. Ultimately, the horse was euthanised for continued dangerous behaviour, which treatment had failed to resolve in the longer term. In conclusion, the presence of cysts in the mandibular condyle and osteoarthritis can be a cause of abnormal behaviour, which may render a horse dangerous to ride. The case report illustrates the complex interaction between the TMJ, behaviour and movement.

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.

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.

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.