This is an open-label, non-controlled prospective pilot study, which included 40 patients diagnosed with knee osteoarthritis (KOA) in the Rheumatology Department of the Hospital Universitari Parc Taulí (HUPT, Sabadell, Spain). Patients were refractory to a previous intra-articular glucocorticoid injection within the past year. Refractory status was defined as the absence of improvement or a decrease in symptoms lasting no more than one month after the glucocorticoid injection. The study comprised two distinct treatment arms, with patients consecutively recruited during outpatient visits at the same clinic. The initial 20 patients received three separate intra-articular injections of Autologous Cytokine-Rich Serum (SARC), administered one week apart. The subsequent 20 patients received a single-dose regimen of SARC injection. Both treatment arms were monitored over a 12-month period. All intra-articular injections were administered by the same team of rheumatologists.

We enrolled participants aged 50 and above who had a confirmed diagnosis of radiographic KOA, determined through a prior knee X-ray examination conducted before the study baseline. Patients’ KOA severity was classified as grade 2 or 3 according to the Kellgren and Lawrence criteria, excluding patients in other stages. Other inclusion criteria required patients to be refractory to a glucocorticoid intra-articular injection within the past year, exhibiting persistent pain measured by a Visual Analog Scale (VAS) score of 40–90mm in all visits throughout the previous year and in the month preceding inclusion. All patients provided informed consent for the study, and the research protocol received approval from the Ethics Committee at our hospital (Reference Approval number: 2021_5028).

Patients attended either 6 or 8 clinic visits depending on their assigned treatment arm. Following the intra-articular SARC administration, whether in a single or three-dose regimen to the target knee, we conducted five additional study-related visits: at 15 days after the last intra-articular injection and then at 1, 3, 6, and 12 months. During all study visits, a rheumatologist examined the targeted knee and assessed pain and function using the self-administered KOOS subscales, which were reversed to facilitate the interpretation of the results. Moreover, the rheumatologist systematically inquired about any adverse events experienced by the patients.

Treatment response was assessed by measuring the percentage of patients who improved by 20% and 50% in reversed KOOS pain and function scores over one year. Non-responders included withdrawals. Results were collected at various intervals – 15 days, 1, 3, 6, and 12 months – with the primary focus on the one-year outcome. To gain deeper insights from our data, we compared the KOOS score values between baseline and the time points evaluated. Results were compared for single and three-dose regimen groups, to assess the need of additional injections in the administration of the SARC treatment.

SARC, the Autologous Cytokine-Rich Serum, is a derivative of the patient's own platelets and white blood cells, which is administered via intra-articular injection. It is a serum without platelets, cells, or fibrinogen that directly contains a concentration of anti-inflammatory cytokines and growth factors.

The Autologous Cytokine-Rich Serum (SARC) processing was carried out using the medical device QREM CYTOKINE with CE marking MED31489 granted by the Notified Body KIWA Cermet (No. 0476). The product manufacturer is the company Tecnologia Regenerativa Qrem S.L. with facility license number 7096-PS granted by the Spanish Agency of Medicines and Medical Devices (AEMPS).

QREM CYTOKINE is closed and automatic lab-in-a-box system consisting of a centrifugation device (QREM DEVICE) and a sterile disposable single-use kit (KIT QREM CYTOKINE).

QREMDEVICE performs a 9-step centrifugation process ranging from 0 to 1400g. The process takes a total of 36min. At the end of the process, the SARC is obtained.

The KIT QREM CYTOKINE consists of two containers (F and A) joined by a channel containing a valve that allows the transfer of F to A when the QREM DEVICE reaches a specific centrifugal force. Container F is filled with 18mL of peripheral blood from the patient plus 2mL of sodium citrate (anticoagulant). Container A is filled with 20mL of peripheral blood. The filling is done through the luer lock ports of the containers, which also have non-return valves ensuring a closed process. Blood and the resulting SARC are confined throughout the process inside the KIT QREM CYTOKINE.

The initial steps of the centrifugation process allow obtaining a concentrate of platelets and leukocytes in the container F and a primary serum in container A. This primary serum contains autologous activating elements such as thrombin and calcium, as well as growth factors and other cytokines. Subsequent steps include opening the valve that allows the mixture of the platelet and leukocyte concentrate with the primary serum in container A, leading to the activation of platelets and leukocytes, forming a fibrin matrix. The final steps involve breaking the matrix, releasing the SARC. All steps are performed automatically by the QREM DEVICE.

The resulting SARC is collected through the luer lock port of container A and must be administered immediately by the rheumatologist.

On the day of the treatment, the patient visited the Rheumatology Department of our hospital, where they underwent a blood extraction of 38mL following the described protocol. This blood was processed to obtain 10mL of SARC using the QREM DEVICE by a nurse trained to proficiently perform the procedure. The SARC was then handed to the rheumatology clinician, who administered the intra-articular injection under aseptic conditions.

For descriptive purposes, categorical variables were presented as absolute and relative frequencies, while continuous measurements were described using medians, minimum and maximum values. Univariate differences between subject groups were assessed by non-parametric techniques, including Mann–Whitney tests for continuous variables and Fisher's tests for contingency tables. Patients who abandoned the study were treated as non-responders in these analyses, and their baseline questionnaire responses were used as imputed values for missing follow-up data.

Reversed KOOS score values were analysed using mixed-effects linear models, incorporating time points, the number of injections and their interaction as explanatory fixed effects, with patients parameterized as a random effect. Missing KOOS values due to patient withdrawal were imputed from the scores reported at baseline, representing a conservative choice that allows the inclusion of these patients in the analysis while avoiding overestimation of treatment effects. Group means, mean differences between groups, along with their standard errors and 95% confidence intervals (95% CI), were retrieved from the models. Statistical significance was determined using Wald tests derived from the models. Results were visually represented using a stripchart, which included group means and 95% CI retrieved from the models. A significance level of 5% was used in all analyses. All data analyses were performed using R.23

A pilot study enrolled 40 KOA patients, split into two groups: one receiving a single SARC intra-articular injection, and another undergoing a three-dose regimen, with 20 participants in each group. Most patients were women aged 50–88 years (median age: 71) and were predominantly overweight or obese (median BMI: 31). KOA mainly affected grade 3 knees, with even right and left distributions. The single-dose group had older participants (median age: 72 vs. 65) and higher BMI (33 vs. 30) compared to the three-dose group. This group also reported higher baseline pain and disability levels (10- and 9-points median increase, respectively) compared to the one dose group. Over the six-month follow-up, 47.5% completed the study, 50% withdrew due to lack of efficacy, and 2.5% experienced a fatal event unrelated to the treatment (lung carcinoma). Withdrawals were classified as non-responders in the analysis (Table 1).

Overall, 59% of participants (23 patients) reported a 20% improvement in their pain levels after 15 days of treatment, as measured by their KOOS scores (Table 2). This proportion remained relatively consistent at the 1-month follow-up (63%, 24 patients), and gradually declined in subsequent assessments to 46% at 3 months (18 patients) and 37% at 6 months (14 patients). Interestingly, the rate of response at one year remained stable at 36% (14 patients), suggesting a sustained level of improvement comparable to that observed at 6 months (Table 2).

The single-dose group initially showed better response rates at 15 days (63%, 12 patients) compared to the three-dose group (55%, 11 patients). Over time, the response rate for the single-dose group declined more sharply, reaching 32% (6 patients) after one year. Meanwhile, the three-dose group peaked at one month (68%, 13 patients) and experienced a slower decline, maintaining a higher response rate of 40% (8 patients) at the one-year follow-up (Table 2).

Patients’ functional disability improvement followed trends like pain level responses. Initially, the single-dose group showed a higher response rate (53%, 10 patients) compared to the three-dose group (40%, 8 patients) at 15 days. By one month, the rates were nearly equal (one dose: 56%, 10 patients; three doses: 53%, 10 patients) but declined more sharply in the single-dose group over time. At the one-year follow-up, 40% of the three-dose group (8 patients) and 26% of the single-dose group (5 patients) sustained a 20% improvement in functional disability, though differences were not statistically significant (Table 2).

When analysed with more stringent criteria (50% improvement), the single-dose group demonstrated an 11% response rate (2 patients) in pain levels after one year of follow-up, while the three-dose group showed a higher response rate of 25% (5 patients). In terms of functional disability, these proportions were 21% (4 patients) for those treated with a single injection and 30% (6 patients) for the three-injection group (Supplementary Table S1).

The study conducted a quantitative analysis of reversed KOOS score changes over time (Figs. 1 and 2; Tables 3 and 4). Missing values were conservatively imputed from baseline scores to avoid overestimating treatment effects. Fifteen days after treatment, statistically significant decreases in pain and functional disability were observed which were comparable between the single-dose and three-dose groups (pain: 17.1 and 16.8 points decrease, p-values <0.0001; functional disability: 16.4 and p-value=0.0002, 14 points decrease and p-value=0.0011, respectively).

Fig. 1.

Pain perception at baseline and follow-up. The circle-shaped dots show the KOOS pain scores recorded at baseline and at 15 days, one month, three months, six months, and one year after treatment with SARC intra-articular injections. Empty dots indicate response to the treatment, defined as a 20% decrease in the score compared to baseline, while filled dots express basal values or lack of such response in the follow-up. Asterisk-shaped dots indicate values for a patient with missing baseline KOOS values and, hence, with response data not available. Gray dots in the follow-up visits are imputed values from baseline for patients who withdrew due to lack of perceived efficacy (20 patients). The diamond-shaped points and the adjacent segments display the adjusted group means of the scores and their corresponding 95% confidence intervals (95% CI), respectively. Adjusted means and 95% CI were derived from a mixed-effects linear model where time point, number of injections and their interaction were included as explanatory fixed effects, and patients were parametrized as a random effect.

Fig. 2.

Perception of function disability at baseline and follow-up. The circle-shaped dots show the KOOS function disability scores recorded at baseline and at 15 days, one month, three months, six months, and one year after treatment with SARC intra-articular injections. Empty dots indicate response to the treatment, defined as a 20% decrease in the score compared to baseline, while filled dots express basal values or lack of such response in the follow-up. Asterisk-shaped dots indicate values for a patient with missing baseline KOOS values and, hence, with response data not available. Gray dots in the follow-up visits are imputed values from baseline for patients who withdrew due to lack of perceived efficacy (20 patients). The diamond-shaped points and the adjacent segments display the adjusted group means of the scores and their corresponding 95% confidence intervals, respectively. Adjusted means and 95% CI were derived from a mixed-effects linear model where time point, number of injections and their interaction were included as explanatory fixed effects, and patients were parametrized as a random effect.

In subsequent assessments, patients treated with a single SARC intra-articular injection exhibited a decrease in their KOOS scores until their evaluation at 6 months of follow-up, where KOOS scores were no longer significantly different to those reported at baseline. After one year, pain levels and functional disability in these patients displayed decreases of 8.5 (p-value=0.0345) and 8.4 points (p-value=0.0536), respectively.

In contrast, patients treated with three injections continued to experience a decrease in KOOS scores at one month (pain: 24.1 points; functional disability: 23.7 points decrease; p-values <0.0001), increased at three months and stabilized at 6 months of follow-up, ultimately resulting in a final decrease of 13.9 (p-value=0.0004) and 16.4 points (p-value=0.0001) in pain and functional disability, respectively (see Supplementary Tables S2 and S3 for further details).

Throughout the study period and follow-up, the research team did not detect any local or systemic adverse events related to SARC. Furthermore, no reactions after the intra-articular administration were reported. One death occurred in the single-dose arm due to a metastatic lung neoplasm, which was not related to the study treatment or procedures.