Patients were eligible for the study if they met all the following criteria: 1) age between 18 and 65 years; 2) an active lifestyle as a professional or regular sport player, i.e., training at least 2–3 times per week; 3) diagnosis of knee OA (classified as a Kellgren and Lawrence (KL) Grade I-III) (Kellgren and Lawrence, 1957) based on a standing weight bearing knee X-ray performed at screening or within 6 months prior to enrollment; 4) a knee visual analogue scale (VAS) pain score, performed within 48 h before the visit Day 0 ≥ 30 mm but ≤80 mm if receiving any analgesic medications; or a pain score of 40 –90 mm for patients free of any analgesic; the contralateral knee should have had a VAS pain score of <30 mm (the target knee was the one demonstrating the greatest VAS pain); 5) patients must have had all analgesic/anti-inflammatory drugs discontinued for 2 weeks prior to therapy except for acetaminophen; 6) the rescue medication, acetaminophen (maximum dose of 3 g/die), or any other analgesic, had not to be taken within 48 h of any visit. Patients were excluded from the study when either one or more clinically significant condition would interfere in the treatment and assessment of the study, or they underwent a prior surgical intervention involving the target joint, or had a history of allergic reaction to an i.a. HA injection or to acetaminophen, or they were participating in other trials, or had a case of previously identified knee trauma.

The primary study objective was to investigate if a single i.a. injection of Hymovis MO.RE. increased knee function 90 days after treatment. The primary endpoint was the average score of the SP1-SP5 questions of the “function during sport and recreational activity” subscale of the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire (Roos and Lohmander, 2003). Score changes were normalized according to a 0–100 scale, increasing with patient improvements.

The secondary objectives of the study included further assessing the efficacy as well as safety of the single injection treatment with Hymovis MO.RE. 90 days after treatment. Additional efficacy objectives and endpoints included measuring biomechanical changes through GAIT analysis, the effect on pain perception through the VAS, WOMAC-A scores (Bellamy et al., 1988), and the KOOS pain subscale (Roos and Lohmander, 2003), again assessing the effect of treatment on knee function through the WOMAC-C score (Bellamy et al., 1988), and evaluating symptoms, activities of daily living (ADL) and quality of life (QOL) of patients using the corresponding KOOS subscores (Roos and Lohmander, 2003). The study also aimed to assess the requirements for daily rescue analgesic medications (simple analgesics, acetaminophen, the allowed “rescue dose” being acetaminophen 3 g/die) while being treated with Hymovis MO.RE.

GAIT analysis involved assessing mean velocity (m/s) and cadence (step/min) (Davis et al., 1991; Ornetti et al., 2010). The GAIT approach is a non-invasive method to measure body kinematics and kinetics: patients were requested to walk at a self-selected speed along a 10 m level surface after applying on their knee several retroreflective spherical markers to determine the joint centers and segment axis (Davis protocol) (Davis et al., 1991). The walk was repeated five times. GAIT data collection and analysis was performed using the ELITE system (BTS, Milano, Italy), with eight infrared video cameras (TVC, BTS, Milano, Italy) for the acquisition of the kinematic and kinetic variables. Two Kistler platforms (Kistler Instruments, Winterthur, Switzerland) were employed to acquire the ground reaction forces (GRF). (Paoloni et al., 2012; Alviti et al., 2017; Lo Torto et al., 2020). The VAS scale used in the present study was a standard 0–100 mm horizontal line, with 0 indicating no pain and 100 mm indicating the worst possible pain. The WOMAC A (5 questions) pain and the WOMAC C (12 questions) knee function questionnaires were as described by Bellamy and colleagues (1988). WOMAC scores were processed as described in the WOMAC user manual, that is, calculating a normalized score ranging from 0 (worst clinical condition) to 100 (best clinical condition). KOOS scores were also normalized, by calculating a 0 (worst condition) to 100 (best condition) KOOS index, as described in the 2012 KOOS user guide. Safety endpoints included the frequency and type of adverse events, patient withdrawals, and the presence of swelling, tenderness, crepitus, redness, and effusion at the target knee.

Patients were enrolled during a screening visit and underwent i.a. injection within 15 days, this visit was regarded as the study baseline. The injection was carried out through a lateral approach using a 4 ml-pre-filled syringe, corresponding to 32 mg Hymovis MO.RE. Following administration, passive flexion and extension of the knee were briefly performed to allow diffusion of the injected fluid throughout the joint. Patients had to limit movements during the following 24 h. Follow-up visits followed at 30, 60, 90, 135, 180, 270, and 360 days thereafter. Clinical (KOOS, VAS, WOMAC) scores were collected at baseline and at the 30, 90, 180, and 360-day control visits; biomechanical (GAIT) data were recorded at 30-, 90- and 180-days. Data concerning concomitant medications and adverse events were collected at each control visit. The 60, 135, and 270-day control visits were carried out by interviewing the patient over the phone and recording information about concomitant medications and adverse events.

The study sample size, 31 patients, was calculated based on the hypothesis of observing a minimum 15-point KOOS sport and recreational activity subscore (primary endpoint) change at 90 days after injection, compared to baseline, with a standard deviation of 21.1 points (Sakpal 2010), and 10% maximum possible dropout. Calculations were made by assuming that the results of statistical tests had 5% maximum error and 95% power. Categorical variables were reported as percentages; non-normally distributed variables were described by medians and ranges; normally distributed variables were described calculating their mean and standard deviation (SD). To investigate the primary and secondary objectives, t-tests for paired data or Wilcoxon signed-rank tests were used to compare VAS, WOMAC, and KOOS scores collected at each time point with those at baseline. To investigate how scores varied over time, they were also analyzed by means of parametric o non-parametric analysis of variance methods, if they had a normal or non-normal distribution respectively. Score changes were regarded as statistically significant if p < 0.05. All statistical analyses were performed using SPSS version 21.0 (IBM Clinical Software). Descriptive statistics were reported at each time for spatial-temporal gait parameters to evaluate the active functional assessment in patients with knee overuse syndrome. Data were expressed as the number of observations, mean, standard deviation (SD), and median. For each parameter, the comparison between treated and untreated knees was carried out using an analysis of the variance. Alternatively, a non-parametric approach has been proposed, the Wilcoxon Rank-Sum test. This test, based on data ranks, was used to compare GAIT parameters between two independent populations without the assumption of normally distributed data.

A General Linear Model (GLM) approach was also used to compare the difference from baseline, as a dependent variable, treatment group, as a factor, and baseline, as a covariate. Linear Regression analysis was used to evaluate the correlation between GAIT parameters and VAS or a knee-specific questionnaire, developed to assess the patients' opinion about their knee (KOOS, WOMAC-A). The Full Analysis Set (FAS) was used for all statistical analyses and data presentation. Considering that the outliers did not increase the valuable information, the analysis was repeated after removing the outliers from the dataset. The outliers were identified using the Explore procedure. To decide how to handle outliers, the first step used the source dataset and ensured that there were no data entries or tool errors. When a value was identified as an outlier, it was compared to the standardized value. The efficacy analysis was conducted with and without the outliers identified by the Explore procedure. Missing data were not replaced.

We described the relationship between clinical outcomes (scores) and spatio-temporal GAIT parameters, using a correlation analysis, which considers the entire patient cohort. In a linear model, we can judge how well the line fits the data (goodness of fit) by calculating the coefficient of determination (or square of the correlation coefficient) R2, which can be interpreted as the percentage of variance in the outcome variable that is explained by the predictor variable. Furthermore, in a linear regression analysis, it is important to evaluate the regression beta coefficient (β), which can be negative or positive and can have a t-value and a significance of the t-value, associated with each outcome. The β coefficient is the degree of change in the outcome variable for every 1-unit of change in the predictor variable. The t-test assesses whether the β coefficient is significantly different from zero. If the β coefficient is not statistically significant (i.e., the t-value is not significant), the variable does not significantly predict the outcome. If the β coefficient is significant, examine the sign of the β. If the β coefficient is positive, the interpretation is that for every 1-unit increase in the predictor variable, the outcome variable will increase by the β coefficient value. If the β coefficient is negative, the interpretation is that for every 1-unit increase in the predictor variable, the outcome variable will decrease by the β coefficient value.

Thirty-two subjects were screened, and 31 patients were enrolled. All were Caucasian, and 23 (74.2%) were women. Their median age was 49 years (range, 25–65). Twelve patients (38.7%) had Kellgren-Lawrence grade I, 16 (51.6%) grade II, and 3 (9.7%) grade III. Nineteen (61.3%) patients suffered from OA in the right knee, 12 (38.7%) in the left one. The VAS median value at the target knee was 72 mm (range, 43–87 mm) while that at the contralateral knee was 0 for all patients. At the screening visit, all patients were in good clinical status, including 5 (16.1%) who were receiving concomitant therapy for previous, not study-related, medical conditions (post bypass (n = 3), hypertension, flu). Twenty-three patients (74.2%) completed the study. Eight (25.8%) did not, either because they were lost to follow-up (n = 2) or withdrew informed consent (n = 6).

A statistically significant increase (p < 0.001) in the knee function KOOS score, compared to baseline, was detected 90 days after the i.a. injection, as well as at every other time point, confirming the efficacy of the treatment. The functional knee improvement increased through the entire follow-up period of interest (Table 1).

Both cadence and velocity were assessed through GAIT analysis and showed a main improvement at 90 days after injection of Hymovis MO.RE. Cadence increased from the mean baseline value of 103.39 ± 9.07 to 110.95 ± 11.29 (p = 0.005). Significant statistical differences from baseline were observed even at day 30 and day 180 (Figure 1). Velocity increased from the baseline value of 1.10 ± 0.14 to 1.28 ± 0.21 (p = 0.001). Velocity was found to have significantly increased at every evaluation visit, even at day 360 (Figure 2). The VAS scores decreased significantly at all time points compared to baseline (p < 0.001 in all cases) (Table 2). Even the WOMAC-A pain score decreased significantly at all time points compared to baseline (p < 0.001) (Table 2). Difficulty in conducting normal activities related to knee function decreased significantly at all time points (p < 0.001 in all cases) (Table 2).

Pain, symptoms, and QOL measured by the corresponding KOOS subscores showed a statistically significant improvement, compared to baseline, at all-time points (p < 0.001). The subjective assessment of ADL, measured by the ADL KOOS subscore, also improved significantly compared to baseline at all time points (p < 0.001), (Table 2).

Tables 3A–D reported the descriptive statistics at each study time point and the results of statistical comparisons, considering the full cohort, between the changes from baseline (Day 0) for the GAIT parameters grouped by treatment. In particular, a statistical significance with a p-value < 0.05 in the comparison between the two groups “Treated knee” vs. “No-Treated” has been detected for the following parameters: Peak Hip Ab-Adduction Moment, Peak Knee Valgus-Varus Moment, Peak Hip Rotation Moment, Peak of Knee Flex-Extension Moment (Figure 3).

Tables 4A–E reported the descriptive statistics at each study time point and the results of statistical comparisons, considering the cohort without outliers, between the changes from baseline (Day 0) for the GAIT parameters grouped by treatment. In particular, a statistical significance with a p-value < 0.05 in the comparison between the two groups “Treated knee” vs. “No-Treated Knee” has been detected for the following parameters: Vertical Force Area, Peak Knee Valgus-Varus, Peak Hip Rotation, Peak Knee Flex-Extension and Peak Hip Adduction (Figure 4).

A statistically significant correlation between “clinical outcome” vs. “GAIT parameter” has been detected in the “Knee treated” group for the following parameters (Table 5). For the predicted parameter “First Flexor-Acceptance Peak” vs. the outcome variable “VAS” the R2 is 0.202, i.e., around 20% of the variance in the outcome is explained by the predictor, the β coefficient is negative i.e., for every 1-unit increase in the “First Flexor-Acceptance Peak Day 30 change from Baseline”, the “VAS Day 30 change from baseline” will decrease by the beta coefficient value (−0.905), with a p-value of 0.011; for the “Max Knee Valgus-Varus” vs. “VAS” the R2 is 0.177, i.e., around 2% of the variance in the outcome is explained by the predictor, the β coefficient is positive i.e., for every 1-unit increase in the “Max Knee Valgus-Varus Day 360 change from Baseline”, the “VAS Day 360 change from baseline” will increase by the beta coefficient value (0.717), with a p-value of 0.046; for the “Max Knee Valgus-Varus” vs. “KOOS-SPORT” the R2 is 0.15, i.e., around 1.5% of the variance in the outcome is explained by the predictor, the β coefficient is positive i.e., for every 1-unit increase in the “Max Knee Valgus-Varus Day 30 change from Baseline”, the “KOOS-SPORT Day 30 change from baseline” will increase by the beta coefficient value (0.223), with a p-value of 0.032; finally, for the “Range Motion Knee Rotation” vs. “WOMAC-A Pain” the R2 is 0.16, i.e., around 2% of the variance in the outcome is explained by the predictor, the β coefficient is positive i.e., for every 1-unit increase in the “Range Motion Knee Rotation Day 30 change from Baseline”, the “WOMAC-A Pain Day 30 change from baseline” will increase by the beta coefficient value (0.186), with a p-value of 0.028 (Figure 5). Moreover, we conducted an ANOVA analysis to determine if there is a statistical significance difference among the two groups “Treated Knee” vs. “Non-Treated Knee” in the four linear models inspected with the previous correlation analysis (Table 6). We noticed particularly interesting results because a significative correlation in the group “Treated” corresponds to a non-significative (or a non-correlation) in the other group “Non-Treated”. In particular, for the predictor “First Flexor-Acceptance Peak” and the outcome variable “VAS” grouped by treatment, the R2 is 0.202 (for “Treated” group) with a p-value of 0.011 and the R2 is 0.118 (for “Non-Treated” group) with a p-value of 0.059; for the predictor “Max Knee Valgus-Varus” and the outcome variable “VAS” grouped by treatment, the R2 is 0.177 (for “Treated” group) with a p-value of 0.046 and the R2 is 0.01 (for “Non-Treated” group) with a p-value of 0.645; for the predictor “Max Knee Valgus-Varus” and the outcome variable “KOOS-SPORT” grouped by treatment, the R2 is 0.15 (for “Treated” group) with a p-value of 0.032 and the R2 is 0.012 (for “Non-Treated” group) with a p-value of 0.561; finally, for the predictor “Range Motion Knee Rotation” and the outcome variable “WOMAC-A PAIN” grouped by treatment, the R2 is 0.157 (for “Treated” group) with a p-value of 0.028 and the R2 is 0.082 (for “Non-Treated” group) with a p-value of 0.118. No severe adverse events (SAE) were reported during i.a. injections or in the follow-up period. Six (19%) patients experienced six adverse events ranging from mild to moderate, all of which were considered unrelated to the investigational product. Two patients had flu, two patients had road accidents, one experienced abdominal pain and one had sternoclavicular pain. All patients who experienced adverse events recovered completely, except one who was lost at the follow-up. Acetaminophen was administered as rescue medication to three patients during treatment with a median daily dose of 1,000 mg for 2, 8, and 15 days, respectively, (Figure 6).

The main limitation of the present study is its small sample size and the lack of a similar control group undergoing the standard treatment. Moreover, only three patients had a grade III KL OA and a surface EMG was not used. Furthermore, no specific questionnaires exploring sports performance were administered to patients, thus allowing only for an indirect assessment of effects specifically concerning sports activity. This calls for large prospective cohort studies to further assess the efficacy of this treatment in regular sports players affected by knee OA. Given the clinical characteristics and expectations of this particular patient population, future studies are also advised to evaluate the possible preventive role of i. a. Hymovis MO.RE. injection in reducing knee injuries as well as its effectiveness when associated with physical rehabilitation. Further investigations should include GAIT assessment, given it may provide objective confirmation of subjectively-measured scores, and it has been shown to be predictive of OA progression (Favre and Jolles, 2017).