Eligibility criteria

To be enrolled in this trial, the following eligibility criteria, assessed at screening, will be met:

Inclusion criteria:

Patients aged 18 years and older with a clinical diagnosis of KOA according to the ACR criteria, with idiopathic/primary knee osteoarthritis classified as Stage II and III according to the Kellgren & Lawrence (KL) grading system, will be evaluated for inclusion to the study, along with the following criteria:

• Pre-treatment pain rating of 4 points or higher on the VAS
• Indicated for intra-articular hyaluronic acid injection
• Previously received conservative treatment for knee osteoarthritis and did not achieve an adequate response (conservative treatment: exercise, non-steroidal anti-inflammatory drugs, physical therapy)
• Body mass index (BMI) between 20 and 40 kg/m²
• Able to provide written informed consent
• Not pregnant or breastfeeding
• At least two years postmenopausal or surgically sterile or fertile but willing to use acceptable methods of contraception
• Patients who agree to complete the washout period for non-steroidal anti-inflammatory drugs prior to the study procedures and administration of the study treatment (this means not taking any NSAIDs or other painkillers for at least 48 hours prior to visits where responses to the study treatment will be assessed).
• Able to walk independently without the aid of walkers, canes, crutches, or other assistive devices,
• With sufficient mental function to understand and correctly answer the questionnaires and scales used to evaluate treatment response (Mini Mental Test score above 24)
• Those for whom the DN-4 questionnaire questions can confirm that the pain is not neuropathic in origin,
• For those with bilateral KOA: provided that the VAS pain scores for both knees are similar (difference < 20).

Exclusion criteria:

• Pregnant women, breastfeeding women, and women planning to become pregnant within one year
• Patients diagnosed with autoimmune or inflammatory rheumatic diseases such as rheumatoid arthritis, gout, pseudogout, psoriasis, SLE, fibromyalgia
• Patients with active inflammation or infection
• Patients using anticoagulant therapy
• Patients with known allergies to hyaluronic acid and other excipients
• Other joint diseases that may interfere with the observation of treatment efficacy
• Other intra-articular injection treatments such as steroids, PRP, or stem cell injections within the previous 6 months prior to inclusion in the study
• Use of aspirin, acetaminophen, or other non-steroidal anti-inflammatory drugs within 48 hours prior to the study treatment, or use of other opioid, cannabinoid, and pyrazolone derivative analgesics
• Intra-articular hyaluronic acid injection within the 6 months prior to inclusion in the study
• Open surgical intervention on either knee within the last year
• Advanced osteoarthritis (KL Stage III-IV) in the hip joint
• Other treatments or practices (alternative medicine, nutritional supplements, etc.) that could produce results conflicting with the study treatment and procedures
• Peripheral neuropathy, vascular insufficiency, hemiparesis, systemic bleeding disorders
• Skin diseases or infections in the knee area where the injection will be administered
• Having osteoarthritis secondary to systemic metabolic diseases, or other painful musculoskeletal diseases
•  > 20 ml of synovial fluid during aspiration during the injection
• Those with varus or valgus deformity greater than 10 degrees or joint motion restriction greater than 10 degrees
• Patients receiving corticosteroid or other immunosuppressive drug therapy at a dose higher than 5 mg prednisolone daily
• Patients whose cognitive functions are insufficient for the assessment of outcome measures (patients with a mental score lower than 24 on the Mini Mental State Examination)
• Patients with planned permanent relocation outside the city or country, major surgery, incarceration, military service, quarantine, etc., for the 12 months during which study follow-ups will take place

Randomization and interventions

Randomization and treatment allocation were generated before participant enrollment using a computer-generated stratified allocation list prepared in Microsoft Excel. Stratification was based on Kellgren–Lawrence grade (II vs III) and disease laterality (unilateral vs bilateral), resulting in four strata. Allocation codes were stored in a password-protected master list accessible only to Investigator-1, the unblinded investigator responsible for treatment preparation and administration. Treatment assignments were placed in sequentially numbered opaque sealed envelopes. Following confirmation of eligibility and informed consent, the participant’s study number was matched with the corresponding treatment code, and the envelope was opened by Investigator-1 immediately prior to treatment preparation. Outcome assessors and participants remained blinded throughout the study. The participants were assigned to one of the following groups:

• Group A: intra-articular hyaluronic acid (Semical Gel-B Cross, 90 mg/3 ml, cross-linked formulation).
• Group B: intra-articular isotonic saline (0.9%, 3 ml).

At the 3-month assessment, following the initial response evaluation, patients will be crossed over to the alternative treatment arm: those who initially received hyaluronic acid will be given isotonic saline, and vice versa. This procedure will also be performed by the unblinded research assistant, with careful attention paid to maintaining blinding. As a result, all patients will receive both treatments during the study period, ensuring that each participant has access to hyaluronic acid treatment in either the first or second injection.

To minimize the risk of unblinding, outcome assessors were fully separated from treatment preparation and injection procedures. Participants were not allowed to observe syringe labeling, preparation, or handling. Allocation information remained accessible only to Investigator-1 and the manufacturer. Outcome assessors will not be present during injection preparation or administration and will not have access to the allocation list, envelopes, syringe serial numbers, or treatment-preparation records. A single-injection regimen of 90 mg of cross-linked hyaluronic acid was selected for this study for two primary reasons. First, the use of a higher-dose formulation allows the evaluation of the long-term effectiveness and duration of therapeutic benefit over a 12-month follow-up period, providing valuable information on sustained clinical outcomes after a single intra-articular administration.

Second, the selected formulation is a highly cross-linked hyaluronic acid product. Cross-linking agents such as BDDE (1,4-butanediol diglycidyl ether) are commonly used in HA-based medical products, and residual cross-linker content represents an important quality-control and biocompatibility parameter. Therefore, this formulation was considered appropriate for systematic monitoring of local tolerability, hypersensitivity-type reactions, and other potential adverse events associated with highly cross-linked HA products.

For participants with bilateral knee osteoarthritis, both knees received the allocated intervention. However, efficacy analyses were performed using a pre-defined index knee. The index knee was defined as the more symptomatic knee at baseline. If symptom severity was identical, the knee with the higher radiographic grade was selected; if radiographic grades were also identical, the right knee was designated as the index knee. All WOMAC-based efficacy analyses and knee-specific outcome assessments were referenced to the index knee.

Study procedure and time points

All study related activities will be performed at the outpatient clinics of Physical Medicine and Rehabilitation department of a tertiary level hospital.

Participants were asked to discontinue nonsteroidal anti-inflammatory drugs and other analgesics for knee pain (except paracetamol) from 2 days before baseline assessment through 12-month follow-up. Paracetamol (up to 3000 mg/day) will be allowed to use as rescue pain relief during the study.

Prior to randomization, screening and baseline assessments were performed. Patients underwent clinical assessment and laboratory testing, including a complete blood count, erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, and a urine test for premenopausal women to rule out pregnancy. The classification regarding the degree of osteoarthritis was performed by analyzing the radiograph of the affected knee/knees according to the method of KL.

All patients will undergo an intra-articular injection at baseline. A safety assessment visit will be conducted between day 3 and day 10 to monitor for early adverse events.The first efficacy evaluation will take place at 3 months post-injection, which will serve as the cross-over point. At this visit, patients initially randomized to HA will receive placebo, and those initially randomized to placebo will receive HA. 3–10 days after the second injection, another safety visit will be performed. Further follow-up assessments will occur at 6, 9, and 12 months to evaluate the persistence of treatment effects. Study timeline is outlined in Fig 1.

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Fig 1. Participant timeline: Schedule of enrollment, interventions, and assessments.

Abbreviations: CRP: C-reactive protein; ESR: eritrosit sedimentation rate; HA: hyaluronic acid; RF:rheumatoid factor; SF-36: short form-36; VAS: visual analogue scale; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index.

https://doi.org/10.1371/journal.pone.0353120.g001

Outcome measures

The primary efficacy endpoint of the study is the between-group difference in WOMAC pain score at the 3-month assessment, before cross-over, using the index knee and adjusted for baseline WOMAC pain score and stratification factors. Secondary outcome measures include pain scores measured by visual analogue scale (VAS) during rest and activity; WOMAC functional score; muscle strength assessed by myometer; functional measures such as the 5 times sit-to-stand test and 6-minute walk test; quality of life assessed by SF-36, Outcome Measures in Rheumatology Clinical Trials (OMERACT) – OARSI responder index, analgesic usage and rate of adverse events.

Data management

All data will be collected prospectively and stored in a secure, password-protected electronic database accessible only to authorized study personnel. Each participant will be assigned a unique study identification number to ensure anonymity. No personally identifiable information will be included in the analytic dataset.

Upon completion of the study and publication of the results, the fully anonymized dataset and corresponding metadata will be uploaded to a public open-access repository.

Statistical analysis

All statistical analyses will be performed using the Statistical Package for the Social Sciences (SPSS) software, version 20.0 (IBM Corp., Armonk, NY, USA) and/or an equivalent validated statistical software package if required for model-based analyses. Descriptive data will be presented as means ± standard deviations or medians (interquartile ranges) depending on the distribution of the variable for continuous variables. Categorical variables will be described with frequency (percentage).The normality of continuous data will be tested using the Kolmogorov–Smirnov or Shapiro–Wilk tests.

The primary efficacy analysis will be based exclusively on the first treatment period (baseline to Month 3), prior to cross-over. The primary endpoint will be analyzed using a linear mixed-effects model (LMM), with WOMAC pain score as the dependent variable, treatment group and time as fixed effects, and participant as a random effect. Baseline WOMAC pain score and stratification factors (Kellgren–Lawrence grade and unilateral/bilateral disease status) will be included as covariates.

Longitudinal secondary analyses including post-cross-over assessments will be considered secondary and exploratory and will be performed using LMMs accounting for repeated measures within participants.

Missing data will be examined for extent and pattern. If missingness is limited, LMM analyses will be performed using all available observations under the missing-at-random assumption. If substantial missingness is observed, appropriate multiple-imputation methods will be considered as sensitivity analyses.

The primary analysis will follow the intention-to-treat (ITT) principle, including all randomized participants in the sequence to which they were originally assigned, regardless of protocol adherence, treatment completion, or cross-over participation. A per-protocol (PP) analysis will also be conducted as a secondary sensitivity analysis, including only participants who completed the first treatment period without major protocol deviations. The consistency between ITT and PP analyses will be examined to assess the robustness of treatment effects.

Sample size was determined a priori using G*Power 3.1.9.2 (Heinrich-Heine-Universität Düsseldorf, Germany) for the first-period parallel-group comparison. Assuming a standardized moderate effect size of 0.5 for the between-group difference in WOMAC pain response at Month 3, a two-sided alpha error of 0.05, and 80% power, 51 participants per group were required, resulting in a total sample size of 102 participants. This calculation was based on a standardized effect size rather than a specific WOMAC pain point difference or formally defined minimal clinically important difference.