Intra-articular injection of two different doses of autologous bone marrow mesenchymal stem cells versus hyaluronic acid in the treatment of knee osteoarthritis: multicenter randomized controlled clinical trial (phase I/II)

This is a phase I/II randomized clinical trial with active control conducted between August 2012 and October 2014, involving the Clínica Universidad de Navarra (Pamplona, Spain) and IBSAL-Hospital Universitario de Salamanca (Salamanca, Spain). All the procedures were approved by the Institutional Review Board of Navarra and the Spanish Agency of Medicines and Medical Devices (Nº EudraCT: 2009-017624-72, Clinical Trials.gov identifier: NCT02123368). All participants provided written informed consent.

Inclusion criteria were as follows: males and females aged 50–80, diagnosis of knee OA according to American College of Rheumatology criteria, visual analogue scale (VAS) joint pain ≥2.5, Kellgren–Lawrence radiological classification scale ≥2, body mass index between 20 and 35 kg/m [2], and availability to be followed during the study period; exclusion criteria were: previous diagnosis of polyarticular disease, severe mechanical extra-articular deformation (>15° varus/15° valgus), systemic autoimmune rheumatic disease, arthroscopy or intraarticular infiltration in the last 6 months, chronic treatment with immunosuppressive or anticoagulant drugs, corticosteroids treatment in the 3 last months, nonsteroidal anti-inflammatory drugs therapy in the last 15 days, bilateral knee OA requiring treatment in both knees, poorly controlled diabetes mellitus, blood dyscrasias, and allergy to HA or bird proteins.

Participants were assigned to comparison groups by an unblinded computer-generated list, based on unrestricted randomization, which was maintained centrally by staff with no clinical involvement in the trial so no center knew the treatment allocation of any patient until the patient had been recruited into the trial.

Three groups were created:

Control group, constituted by patients who received a single intra-articular injection of 60 mg HA (Hyalone^®) in a final volume of 4 ml.

Low-dose BM-MSCs group, constituted by patients who received a single intra-articular injection of 10 × 10⁶ autologous cultured BM-MSC in 1.5 ml Ringer’s lactate solution, followed by an intraarticular injection of 4 ml HA.

High-dose BM-MSCs group, constituted by patients who received a single intra-articular injection of 100 × 10⁶ autologous cultured BM-MSCs in 3 ml Ringer’s lactate solution, followed by an intraarticular injection of 4 ml HA.

We estimated that a sample size of ten patients per group was required to detect an effect size of 0.6 with a power of 80 %, assuming a balanced allocation to treatment groups, and a 5 % type I error probability.

BM-MSCs were generated under good manufacturing practice conditions (GMP) with standard operating procedures. Briefly, bone marrow (100 ml) was harvested from the pelvic bone (iliac crest) under sterile conditions. The mononuclear cell fraction was isolated by Ficoll density gradient centrifugation (Ficoll-Paque, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells, ranging between 20 × 10⁶ and 60 × 10⁶, were subsequently seeded in 175 cm² flasks with growth medium, which consisted of αMEM without ribonucleosides (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 5 % platelet lisate, 2 units/ml heparin, penicillin–streptomycin at 1 % (Gibco) and 1 ng/ml human fibroblast growth factor (bFGF) (Sigma-Aldrich, St. Louis, MO, USA). The flasks were maintained in culture at 37 °C in 5 % CO₂ atmosphere. The growth medium was changed every 3–4 days. About 10–15 days later, colonies were formed and the cells were split with TrypLE Select™ (Life Technologies) and seeded at 3000–5000 cells/cm². Once 70–80 % confluence was reached, cells were split again and cultured until they were available at the amounts required to be administered to patients. Finally, cells were harvested with TrypLE Select™, washed three times with PBS and resuspended in Ringer’s lactate buffer (Grifols, Barcelona, Spain) containing 1 % human albumin (Grifols), to be administered within 24 h of harvesting of the cells. Cells were characterized according to ISCT criteria. Cells were then analyzed by flow cytometry (FACSCalibur, BD Biosciences, San José, CA, USA) with the appropriate antibodies (BD Biosciences) to confirm expression of surface markers CD90, CD73 and CD44, as well as absence of CD34 and CD45.

Cell injection was performed without radiographic guidance through a lateral patellar approach by three different orthopaedic surgeons from both involved centers (Additional file 1: Figure S1), 3–4 weeks after the iliac crest biopsy had been performed. In 90 % of the patients, cells were administered within the first hour after being harvested. For this purpose, a 19 G needle was used in two consecutive intraarticular injections. In the first one, 10 × 10⁶ (low dose) or 100 × 10⁶ (high dose) BM-MSCs were administered in 1.5 and 3 ml doses respectively. Subsequently, 4 ml HA (Hyalone^®) were injected using the same via.

The occurrence of complications and/or adverse effects during the study was registered. In addition, the response to the intra-articular infusion of HA with or without BM-MSCs was assessed using the following procedures:

A goniometer-based evaluation of the articular range of motion at baseline i.e. before treatment administration, and 3, 6 and 12 months after treatment.

Two scale-based methods Visual Analog Scale (VAS) [25] and the Likert version of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) [26, 27], evaluated at baseline and 3, 6 and 12 months after treatment, to clinically assess pain and function. VAS ranges from 0 (maximum relief, i.e., no pain) to 10 (no relief, i.e., maximal pain). WOMAC comprises three subscores: pain, which includes 5 items; stiffness, with 2 items; and physical function, with 17 items. According to previous literature, patients were considered WOMAC responders when they reported an improvement of 20 % in at least two items together with an improvement of ten points in the overall scale [28].

Rosenberg X-ray projections at baseline and 6 and 12 months afterwards to provide a radiographic assessment of the joint space width. A custom methacrylate patient positioner was used to achieve a comparative view (Additional file 2: Figure S2).

A magnetic resonance imaging (MRI) study at baseline and 6 and 12 months after treatment. Two experienced radiologists evaluated MRI images in a blinded manner by assessing the number and location of the lesions, cartilage thickness, signal intensity, and subchondral bone alteration and volume, following the Whole-Organ Magnetic Resonance Imaging Score (WORMS) protocol, in which higher score values indicate more damage [29]. 3T Magnetom TRIO equipment (Siemens, Erlangen, Germany) was used following a protocol which included an axial T1 weighted image (WI) with slice thickness of 5 mm, coronal T1 WI (4 mm), sagittal T1 WI (4 mm), sagittal T2 FS WI (4 mm) and sagittal gradient echo 3D (DESS) (2 mm).

The analyses were performed according to treatment assignment, and all available data from all patients were included in the analyses, following the intention-to-treat principle. Descriptive data summaries are presented as median [interquartile range (IQR)] or percentages. Within each group, the comparison of each clinical and radiographic endpoint between the value obtained at 6 or 12 months and the baseline value, i.e. the one obtained immediately before the administration of the treatment, was performed using the Mann–Whitney U test. Changes in the same end points over time were determined calculating the differences between the measurements collected at the 6 or 12-month follow-up visit and the baseline visit. Subsequently, comparisons between treatment groups were carried out using the Kruskal–Wallis test and the Mann–Whitney U test. All tests were two-tailed. A p value of 0.05 was considered to indicate statistical significance, without adjustment for multiple testing. All analyses were performed using Stata 14 (StataCorp. 2015. Stata Statistical Software: Release 14. College Station, TX: StataCorp LP) and IBM SPSS Statistics 20 (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp).

Thirty-two patients were assessed for eligibility, and were consecutively randomized to treatment groups (Fig. 1). Two patients who had been randomly assigned to the control group withdrew consent and were excluded from the trial. All the groups showed similar baseline characteristics of age and body mass index. Patients in the three groups showed an uneven distribution according to the Kellgren–Lawrence scale but without statistical significance (p = 0.585, Table 1).

No serious adverse events or complications derived from the procedures or treatments were noted. There were no clinically important trends in the results of physical examination, vital signs and laboratory tests during the study. Articular pain requiring anti-inflammatory treatment during the first 24 h after infiltration was observed in 1, 3 and 6 patients in the control, low-dose BM-MSC and high-dose BM-MSC groups respectively. All patients recovered completely without sequelae and no treatment group-dependent differences were detected in the dose of required anti-inflammatory drug or in the time that passed until recovery.

VAS and WOMAC clinical scores were used in order to obtain the best picture of how patients perceived their own evolution. Evaluations were performed before the administration of treatment and 3, 6 and 12 months afterwards, and the results are summarized in Fig. 2, Additional file 3: Table S1 (VAS) and Table 2 (WOMAC). The patients that were solely given HA did not show changes during follow up in their pain status according to VAS (Fig. 2; Additional file 3: Table S1). Furthermore, although they initially perceived some improvement according to the WOMAC pain and physical function subscores, this perception was not significantly sustained in the long term (Table 2). Inatraarticular delivery of BM-MSCs, specially when used at high dose, enabled patients to perceive an improvement in their perception of pain in their daily activity. On one hand, the VAS score value was significantly reduced upon treatment with low and high BM-MSC doses at all follow-up times (Fig. 2; Additional file 3: Table S1). Furthermore, treatment with 100 × 10⁶ cells was associated with a significant improvement in all WOMAC subscores at 12 months (Table 2). It is important to note that, when the overall WOMAC value at 12 months was subtracted from the baseline value in each patient, the median decrease in the score, i.e. the relief of the symptoms, was notably larger if patients had been treated with BM-MSCs [−6.5 (−19, 4), −14 (−27, 4), and −14 (−15, −8), median (IQR), for control, low-dose and high-dose BM-MSCs groups respectively]. Thus, only the patients who had been treated with BM-MSCs met criteria to be considered WOMAC responders in the long term.

The knee flexion and extension ranges of motion were significantly improved in the patients who were treated with BM-MSCs and the effect was seen earlier in patients receiving the higher doses of BM-MSC. No improvement was seen in patients receiving HA alone (Fig. 3; Additional file 4: Table S2).

The analysis of the knee joint space by X-rays during follow up showed a borderline reduction in the control group (p = 0.05 at 12 months), which was not observed in patients treated with high dose BM-MSC (Table 3; Additional file 5: Table S3). The assessment in the low dose group was not possible because the baseline value was 0. These results suggest that BM-MSC may halt the progressive loss of cartilage observed in patients with OA despite the use of HA.

Consistent with the X-Ray results, the analysis of the MRI following the WORMS protocol showed a reduction in the score value during follow up (Table 4). Patients treated with high dose BM-MSCs showed a median improvement of 4 points at 12 months, with an improvement of 22 points in 25 % of patients, while there were no signs of improvement either in the control or in the low BM-MSC group.