Results: Primary Endpoints
As primary endpoints we choose the structural elements of histology and gene expression of constitutive cartilage extracellular matrix and those genes that relate to catabolic activity.
Histological Evaluation
Histological evaluation of samples from three regions of interest (lesion, kissing region, opposite facing cartilage to lesion, and control; Fig. 2A) showed significant improvement in several metrics as a result of sprifermin treatment.
In lesion, all placebo, HA and sprifermin treated groups showed a fibrotic non-cartilaginous repair tissue and their overall assessment was significantly deteriorated (Fig. 2D). We detected most marked changes in the kissing region where both 10 g and 100 g sprifermin groups showed an improvement in repair tissue (Fig. S1).
In the kissing region group,100 g sprifermin showed significant improvement in surface architecture (93±4.33 versus 71.34± 26.63, p = 0.021; Fig. S1E), decrease in the lesion intensity (91.94± 11.71 versus 73.48± 25.94, p = 0.015; Fig. S1G) compared to placebo group (scores and the lesion intensity are inversely related), and a nearly to significant improvement in the overall assessment (p = 0.057; Fig. 2E). In the same region, 10 g sprifermin significantly associated with improved surface architecture compared to placebo (sprifermin 93.98 ±4.11versus placebo 71.34 ±26.63, p = 0.009; Fig S 1E) and five out of six animals showed improvement in overall assessment when compared to placebo (Fig 2E). For the kissing region group in the 30 g sprifermin group, there was an opposite trend detected (p<0.05) with reduced cellularity, increased clustering, and disrupted surface architecture when compared to sham joints (Fig. S1).
In the control region group, except for a significant decrease in the overall assessment (Fig. 2C) and cellularity scores following treatment with 10 g sprifermin, and a decrease in surface architecture score in 30 g group, other metrics for 10 g and 30 g groups and all metrics for 100 g group were comparable with sham. In addition, in lesion, kissing region, and control groups, sprifermin treatment resulted in an increase in the average cell number. This increase was significant in the lesion of the 10 g group; Fig. S1, K to M).
Gene Expression
We isolated RNA and evaluated gene expression in three regions: lesion, peri-lesion, and kissing region (Fig. 2A). In peri-lesion group receiving 100 g sprifermin, there was a significant increase in aggrecan (p<0.05) and COMP (p<0.01) expression compared to HA and placebo groups (Fig. 3A and C). In the same treatment group, the average expression of collagen type II increased (100 g sprifermin 4885±3053 versus placebo 3031± 2672) and 5 out of 6 animals showed an improvement in collagen type II expression when compared to the average gene expression in placebo and HA groups (Fig. 3B). In the case of collagen type I expression, we detected a significant decrease compared to HA and placebo treatment groups (Collagen type I 1.068±0.57 versus HA 11.78± 8.29 and placebo 20.64± 27.63, p = 0.0058 and p = 0.018 respectively; Fig. 3D).
In kissing region group, we detected a dose dependent increase in average aggrecan, collagen type II and COMP expression with highest expression in 100 g (Fig. 3E to G). In 100 g sprifermin, COMP expression showed a near to significant (p = 0.055) and significant (p = 0.018) increase in expression compare to placebo and HA treated joints. Although we did not detect any significant changes in collagen type I expression, all animals except one showed a decrease in gene expression compared to the average of the gene expression in placebo and HA groups (0.0578 and 1.001 respectively; Fig. 3H).
In 100 μg sprifermin treated lesion group, when compared to placebo, there was an increase in level of aggrecan, collagen type II and COMP expression and a decrease in level of collagen type I expression, however these changes were not significant (p = 0.453, 0.922, 0.103, and 0.136 respectively; Fig. 3I to L). Five out of six animals treated with 100 μg sprifermin showed an improvement (reduction) in collagen type I expression compared to the average gene expression in placebo and HA groups (average gene expression 38.9 and 31.32 respectively; Fig. 3L). Regarding the control cartilage samples, there was no relative changes in gene expression compared to sham, with the exception of an increase in collagen type II expression in 30 g and 100 g sprifermin (p = 0.001 and p = 0.055 respectively; data not shown).
Results: Secondary Endpoints
In order to assess relevant clinical measurements and more functional outcomes, we performed as secondary endpoint measurements the following: clinical assessments including lameness and flexion tests, gross joint evaluations, synovial fluid analysis, arthroscopic and radiographic imaging.
Clinical Assessments
All animals recovered from general anesthesia without incident. One animal (Horse # 4) had an episode of abdominal discomfort after second look arthroscopy that required surgical management. She subsequently developed grade 2 gastric ulcers and required omeprazole therapy thereafter for one month. Horses #8 and #17 were lame enough post-operatively that a longer period stall rest and phenylbutazone were given.
For lameness and flexion assessment, looking at week by week results and combining scores from all sprifermin treated animals, scores peaked in the early postoperative phase and again after the second look arthroscopy at week twelve. This secondary peak was more prominent in placebo treated group (Fig. S2A and B). In addition, comparing sprifermin to HA treatment, scores from sprifermin treated joints were closer to sham values (Fig. S2A and B). We observed a gradual improvement in lameness and flexion test scores and at time points after week 14, improvement in scores reached statistical significance (p<0.05; Fig. S2C and D). When we analyzed the lameness sum scores for the whole duration of this study, it was seen that no single dose of sprifermin was superior to placebo treatment (Fig. 4A), but the combined results of three doses of sprifermin showed a significant improvement in lameness scores (2.5 ± 3.777 and 6.30 ± 5.51, p = 0.046; Fig. 4B). In the matter of flexion test, we observed a near to significant improvement when we combined results from various sprifermin dose treatment groups (sprifermin 3.389± 6.48, placebo 7.667± 6.851, p = 0.053; Fig. 4D).
Synovial Fluid Analysis
Synovial fluid was obtained from all horses at all time points without complication. There was no effect of treatment with sprifermin, HA or placebo on total synovial fluid white cell counts or total protein (Fig. S2E and F).
Second Look Arthroscopy and Gross Evaluation
Twelve weeks post-surgery, arthroscopic evaluation demonstrated a significant improvement in cartilage defect healing in joints treated with sprifermin compared to joints treated with HA or placebo (sprifermin 10.19± 2.71 versus placebo 9.16 ±2.93, p = 0.02, Fig. 5B). There was no significant difference between groups when looking at individual doses of sprifermin or between HA and placebo treated groups (Fig. 5A). Evaluation of sub-scores showed significantly superior scores in surface texture (p = 0.05) of sprifermin treated joints (Fig. S3F). In addition, treatment with 10 g, 30 g or 100 g sprifermin demonstrated a higher benefit when compared to HA treatment (37.6%, 24.7% and 34.1% respectively versus 17.6%).
Post-mortem evaluation revealed that treatment with no single dose of sprifermin resulted in a significant improvement in overall gross morphology (Fig. 5C). When we combined results from various sprifermin treatment groups, a near to significant improvement was detected (22.42±3.40 versus 20.03±3.09, p = 0.060; Fig. 5D). In analysis of multiple sub-scores, we detected a significant (p = 0.039) and a near to significant (p = 0.058) improvement in cartilage surface and defect fill respectively (Fig. S 4I and K).
We detected no significant difference between HA and placebo treated limbs. The percentage benefit evaluation showed a larger and inversely dose related benefit in sprifermin treatment compared to HA (ranged from 37.4% to 21.4% versus 0.53%).
Radiographic and Computed Tomography Scores
Radiographs and CT examination showed decreased osteoarthritic changes in sprifermin treated joints compared to placebo and HA (Fig. 6).
At week 12, radiography scores were significantly improved in high dose (100 g) sprifermin (p<0.05) or when we combined the scores from three sprifermin dose-treatment groups (p<0.01; Fig. 6B and C). The same significant improvement in radiographic scores of the 100 g group was detected at week 24 post-surgery (p = 0.016; Fig. 6D) and combined sprifermin scores were also significantly improved (8.31 ±0.941 versus 17.39 ±0.883, p = 0.011; Fig. 6E). Peri-articular soft tissue swelling and soft tissue mineralization were the main radiographic features improved as a result of sprifermin treatment (p = 0.024 and 0.064 respectively (Fig. S5E and F). There was an increased benefit in low dose (10 g) and high dose (100 g) sprifermin compared to HA (48.6% and 70% respectively versus 25.7%).
At week 24, CT scores of combined sprifermin showed significantly less osteoarthritic change compared to placebo (20.39± 2.38 versus 18.53±2.09, p = 0.043; Fig. 6H). Evaluation of sub-scores showed significantly reduced osteophyte development in sprifermin treated limbs at 24 weeks (p<0.01) compared to HA and placebo treated joints (Fig. S 6G). Additionally, treatment with any of three doses of sprifermin had a larger benefit when compared to HA (ranged from 26.3% to 36.4% versus 3.3%).