1. Horses and study design
The study protocol was approved by the ComEth Anses/ENVA/UPEC (protocol code 21–037#30112 OA-ACTIVE). A power analysis was performed considering an alpha of 0.05 and an actual power of 0.95. Since no detailed information on the primary outcome of this treatment in horses was available, the power analysis was based on synovial effusion score data obtained from a previous study evaluating the effects of another biological therapy in normal equine joints (20). Based on the analysis, seven fetlocks were required per treatment group. To ensure the exact number in case of a major health problem causing exclusion of a horse from the study, 8 clinically healthy horses owned by the Center of Imaging and Research on Equine Locomotor Pathology (CIRALE) were included. There were 5 geldings and 3 mares, aged from 12 to 28 years old. General clinical and locomotor evaluations were performed before inclusion on each horse, and all underwent ultrasonographic and radiographic examinations of both front fetlocks to rule out the presence of preexisting disease.
The timeline of the study design is represented in Fig. 9. On day (D) 0 (D0; baseline), all horses included in the study were randomly administered 2.4 mL of functionalized nanogels in one metacarpophalangeal (fetlock) joint and 2.4 mL of 0.9% saline solution in the contralateral metacarpophalangeal joint as a negative control. A uniform distribution of the nanogel and saline as negative control was split between left and right fetlocks. A trained operator blinded to the contents of the injected substance (LB) performed all injections after aseptic preparation of the skin and sedation (intravenous administration of a combination of 0.01 mg/kg detomidine and 0.01 mg/kg butorphanol). Two overdosing conditions were tested: overdose by repeated weekly injections of the therapeutic dose and overdose by a single injection of thrice the therapeutic dose. Therapeutic dose injections were performed once weekly for 3 weeks (D0, D7, D14) using a lateral approach on the flexed limb, with a 20-gauge needle inserted between the metacarpal condyle and the articular surface of the lateral proximal sesamoid bone. Injection of the triple dose was performed on D21 using the same approach.
All horses were confined to 3.5 x 4 m stalls from the day of each injection until 3 days after injection and then returned to the paddock. No bandages or medications were used to avoid any interference with the inflammatory response. The vital parameters and appetite of the horses were monitored twice daily to check for signs of discomfort.
Clinical and ultrasonographic follow-up of the front fetlocks were performed on each injection day prior to injection, one, three and seven days after each injection, and 14 days after the last joint injection (D35) for short-term evaluation. A medium-term follow-up was finally performed three months after the first injection (D84). Synovial fluid samples (1 mL) were obtained from each fetlock joint on D0 (before injection), 7 days after each injection and on D84 using the same technique used for injections and during the same procedure on the corresponding days (D0, D7, D14, D21): the same needle was used, only the syringes were changed, and synovial samples were collected before injection. The samples were divided into EDTA and dry tubes.
In the case of a major inflammatory reaction leading to clinical grades ≥ 3/4 (or ≥ 3/5 for lameness), investigators were allowed to administer non-steroidal inflammatory drugs to relieve pain and excessive inflammation. This deviation from the protocol excluded the horse from the study, from the time point of drug administration onward. All methods performed in this study were carried out in accordance with the ARRIVE guidelines.
2. Preparation of functionalized nanogels for injection
Two formulations of functionalized nanogels were combined according to the protocol described in our previous study (19). The first formulation (BQ123-CS) was composed of chitosan (CS) conjugated to the peptide BQ-123 (ChinaPeptides Co., Ltd., Shanghai, China), and the second (R954-HA = R) was composed of HA conjugated to the peptide R-954 (kind gift from Pr. Sirois, Québec, Canada). Briefly, peptide grafting was performed according to the EDC/NHS (Sigma‒Aldrich Canada Co., Oakville, ON, Canada) coupling chemistry. The concentration and grafting rates were determined by spectrofluorometry (Hitachi F-2710 spectrophotometer, Hitachi High Technologies America, Inc., Pleeasanton, CA, USA).
An ionic gelation process under aseptic conditions was performed for nanogel synthesis as previously described (9). A combination of a chitosan solution (CS, 2.5 mg/mL), sodium tripolyphosphate (TPP, 1.2 mg/mL) (Alfa Aesar, Ward Hill, MA, USA) and 60 kDa sodium hyaluronate solution (HA, 0.8 mg/mL) was used. Nanogel suspensions were then purified by dialysis and lyophilized with 8% sucrose (Sigma‒Aldrich Canada Co., Oakville, ON, Canada) as a cryoprotective agent before storage at -20°C.
Two to three hours before injection, the frozen nanogel powder was placed at room temperature. One to two hours before injection, 0.05 mL of sterile 5% glucose solution (Laboratories Chaix et du Marais, La Chaussee Saint-Victor, France) were injected under aseptic conditions with an insulin syringe and a 23G needle into the vial containing the freeze-dried nanogels. The vial was vortexed for one minute and allowed to rest for 14 minutes at room temperature. The injection of 0.05 mL of 5% glucose, vortexing and resting was repeated three more times. The diluted vial was then added with 0.2 mL of 5% glucose solution with a sterile insulin syringe. The vial was vortexed for 20 seconds and allowed to rest for 5 minutes at room temperature. These steps were repeated another 2 times, adding 1 mL of 5% glucose solution each time. A final volume of 2.4 mL was obtained. The single dose of the functionalized nanogels contained 1.2 µM BQ-123 and 0.54 µM R-954. The triple dose contained 3.6 µM BQ-123 and 1.6 µM R-954. All injection vials were sterile and endotoxin-free. The therapeutic dose was calculated based on findings from in vitro studies (19), for an average horse weight of 500 kg.
3. Assessments and outcomes
The primary outcome was joint effusion as a clinical sign of joint inflammation. This outcome was assessed blindly by the same experienced operator (SJ) using a five-point scale (20) from normal to severe (Table 1). The secondary outcomes included one ultrasonographic outcome and six outcomes related to synovial fluid analysis.
Table 1
Detailed grading criteria for each clinical and imaging parameter.
| Grade | Swelling | Joint effusion | Passive flexion | Lameness | Subcutaneous edema | Synovitis |
|---|
| 0 Normal | Absent | Concave aspect of the proximo-palmar recess of the metacarpophalangeal joint. No lateral swelling when a medial pressure is applied on the recess. | No reaction | Absent | Absent | Mild amount of fluid in the proximo-palmar recess of the metacarpophalangeal joint. Concave aspect of the skin. No motion of the fluid when pressing on the recess. |
| 1 Mild | Minimal at the level of the joint | Flat aspect of the proximo-palmar recess of the metacarpophalangeal joint. Mild lateral swelling when a medial pressure is applied on the recess. | Reaction to moderate flexion | Difficult to observe, not consistently apparent regardless of circumstance | Mild | Concave to flat aspect of the skin. Mild amount of fluid in the proximo-palmar recess of the metacarpophalangeal joint. Motion of the fluid when pressing on the recess. |
| 2 Moderate | Mild at the level of the joint | Convex aspect of the proximo-palmar recess of the metacarpophalangeal joint. Lateral swelling easy to obtain when a medial pressure is applied on the recess. | Reaction to mild flexion | Difficult to observe at walk or trot in a straight line, consistently apparent under certain circumstances | Moderate | Convex aspect of the skin. Moderate amount of fluid in the proximo-palmar recess of the metacarpophalangeal joint. Motion of the fluid when pressing on the recess. |
| 3 Substantial | Moderate, extending to the cannon bone and pastern | Convex aspect of the proximo-palmar recess of the metacarpophalangeal joint exceeding the suspensory ligament branches. Soft consistency of the recess on palpation. | Reaction to light flexion | Consistently observable under all circumstances | Substantial | Convex aspect of the skin. Substantial amount of fluid in the proximo-palmar recess of the metacarpophalangeal joint and mild amount of fluid on the dorsal recess of the joint. |
| 4 Severe | Severe, up to the carpus or higher | Convex aspect of the proximo-palmar recess of the metacarpophalangeal joint exceeding the suspensory ligament branches with a taught consistency of the recess on palpation indicating synovial pressure. Synovial distension of the dorsal recess of the joint. | Violent retraction of the limb | Obvious lameness | Severe | Convex aspect of the skin. Marked amount of fluid in the proximo-palmar recess of the metacarpophalangeal joint and substantial amount of fluid on the dorsal recess of the joint with synovial pressure. |
Ultrasonographic examinations were performed by the same blinded veterinary specialist (SJ) using a 7.5 MHz linear transducer (Aloka Prosound Alpha 10, Hitachi Healthcare, Twinsburg, OH, USA) and a systematic protocol including transverse and longitudinal scans from the dorsal to the collateral aspects of each fetlock (20). This operator assigned a synovitis score according to the scoring system previously described (20) as the secondary ultrasonographic outcome (Table 1).
Synovial fluid outcomes included measurements of total protein concentration, total nucleated cell count (ADVIA® 120 Hematology System, Siemens) and viscosity scoring (30). The concentrations of PGE2, IL-1β and MCP-1/CCL-2 were also quantified by ELISA (IL-1β: DuoSet ELISA Equine IL-1β/IL-1F2, R&D Systems; PGE2: Prostaglandin E2 Assay, R&D Systems; MCP-1/CCL-2: Equine MCP-1/CCL-2 ELISA Kit, Thermo Fisher Scientific) to assess joint inflammation and cartilage degradation.
Five other clinical parameters and one ultrasonographic parameter were monitored to document the response to the injections but were not included as variables in the statistical analysis. The fetlock circumference at the mid-level of the proximal sesamoid bones and mid-metacarpal circumference (identified by shaved skin landmarks) were measured by the same operator (AT). Both measurements were taken in triplicate and averaged. Second, sensitivity to digital flexion tests and local swelling were evaluated by the same operator (SJ) using the same five-point scale from normal to severe (20) (Table 1). Finally, the degree of forelimb lameness was graded after two 30-meter round trips at the trot on a straight line on a scale of 0 to 5 in accordance with the lameness scale of the American Association of Equine Practitioners (31) by the same veterinarian specialist (SJ). The presence of subcutaneous edema was also recorded on ultrasound and graded using a five-point scale from normal to severe (Table 1).
4. Statistical analysis
Statistical analysis was performed after the data had been visually assessed for normality and homogeneity. The mean and standard deviation of normally distributed outcomes and the median and quartiles of the other variables were calculated at each time point for each treatment group. To test the first two hypotheses, a linear mixed model with treatment and date as fixed effects and horse as a random effect was used. The treatment groups were then compared at selected dates using a Dunnett's test or its nonparametric equivalent for outcomes for which normality and/or homogeneity were not met. Comparisons were made on D1 (first hypothesis), D15 and D22 (second hypothesis) for the primary outcome and the secondary ultrasonographic outcome and on D7 (first hypothesis), D21 and D28 (second hypothesis) for secondary synovial outcomes. To test the last hypothesis, a linear mixed model with treatment and date as fixed effects and horse as a random effect was also used. D7 and D84 were compared to D0 for each treatment separately using Dunnett's test or its nonparametric equivalent for outcomes for which normality and/or homogeneity were not met. All the statistical analyses were performed using R software (version 3.4.3; R Foundation for Statistical Computing, Vienna, Austria) or Excel 2016 for Windows (Microsoft, Redmond, DC, USA). The alpha type 1 error was set at 5%.