This is a phase II randomized clinical trial with active control conducted between August 2016 and July 2018, involving the Clínica Universidad de Navarra (Pamplona, Spain); IBSAL-Hospital Universitario de Salamanca (Salamanca, Spain) and Hospital Vithas San José (Vitoria, Spain). All the procedures were approved by the Institutional Review Board of Navarra and the Spanish Agency of Medicines and Medical Devices (Nº EudraCT: 2011-006036-23, Clinical Trials.gov identifier: NCT02365142) an Ethics Commite for Clinical Research of Navarra, Salamanca and País Vasco. All participants provided written informed consent.
Criteria for eligibility of patients
Inclusion criteria were males and females aged 18–80, 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/m2, previous failed treatment with hyaluronic acid (HA) and availability for follow-up during the study period.
Exclusion criteria were: previous diagnosis of polyarticular disease, severe mechanical deformation (> 15° varus/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, non-steroidal anti-inflammatory therapy in the last 15 days, bilateral knee OA requiring treatment in both knees, poorly controlled diabetes mellitus, blood dyscrasias.
Patients were randomly assigned to receive either PRGF® or BM-MSC and PRGF® treatment with allocation as per a computer-generated randomization schedule. This allocation procedure was centralized to ensure that no center knew the treatment allocation of any patient until the patient had been recruited into the trial. The biopsy linked to the use of autologous cells did not allow any blinding procedure for patients nor clinicians for ethical reasons. Blinding processes were carried out for imaging evaluation and statistical analysis. The control group was constituted by patients who received PRGF® as a weekly single intra-articular injection of PRGF®, final volume of 8 ml, for 3 weeks. The BM-MSCs group was formed 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 8 ml of PRGF®. A weekly single intra-articular injection of PRGF®, in a final volume of 8 ml, was applied for 2 additional weeks.
Sample size calculation
A sample size of 60 patients (30 patients per group) was required to achieve a 95% power to detect a Cohen’s effect size of 1, assuming a standard deviation for both groups of 10, an alpha value of 0.05 (two-sided test), and an expected dropout rate of 10%.
BM-MSCs were generated under good manufacturing practice conditions (GMP) with standard operating procedures as previously described . 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). Mononuclear 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 lysate, 2 units/ml heparin, penicillin-streptomycin at 1% (Gibco, Life Technologies) and 1 ng/ml human fibroblast growth factor (bFGF) (Sigma-Aldrich, St. Louis, MO, USA). The flasks were maintained in culture at 37ºC 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 3,000–5,000 cells/cm2. Once 70–80% confluence was reached, cells were split again and cultured until they were available in 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 hours 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.
PRGF® was obtained with PRGF-Endoret (BTI System II; BTI Biotechnology Institute, Vitoria, Spain) by centrifugation at 580 x g for 8 minutes at room temperature. The top volume of plasma, with a platelet count similar to peripheral blood, was not used. The plasma fraction, located just above the sedimented red blood cells but not including the buffy coat, was collected in another tube. This plasma contains a moderate enrichment in platelets (2- to 3-fold the platelet count of the peripheral blood) with no leukocytes. Thereafter, in the injection room 10% Calcium Chloride was added to activate platelets (0.05 ml of calcium chloride per ml PRP).
Cell and PRP injection
Cell and PRGF® injection were performed through a lateral patellar approach as previously reported . For cell treatment, cell injection was applied 3–4 weeks after the iliac crest biopsy had been performed. In all of the patients, cells were administered within the first hour after harvesting. For this purpose, a 19 G needle was used in two consecutive intraarticular injections. In the first one, 100 × 106 BM-MSCs were administered in 3 ml of Ringer lactate. Subsequently, 8 ml of PRGF® were injected using the same route. In the PRGF®-only group, 3 separate doses were applied, one week each, 8 ml of PRP per injection.
Outcomes of interest
The clinical response to intra-articular infusion of both treatments was assessed using the following procedures:
To clinically assess pain and function, two scale-based methods, Visual Analog Scale (VAS) and the Likert version of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), were evaluated at baseline, 3, 6 and 12 months after treatment [29, 30]. VAS ranges from 0 (maximum relief, i.e. no pain) to 10 (no relief, i.e. maximal pain). WOMAC comprises three sub scores, 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% on at least 2 items together with an improvement of 10 points in the overall scale .
To provide a radiographic assessment of the joint space width, Rosenberg X-ray projections were taken at baseline and 12 months afterwards. A custom methacrylate patient positioner was used to achieve a comparative view as previously described .
A Magnetic Resonance Imaging (MRI) study was carried out at baseline and 12 months after treatment. One experienced radiologist 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 . Three Tesla Magnetom TRIO equipment (Siemens, Erlangen, Germany) was used following a protocol which included an axial T1 weighted spin-echo (SE) (TR 700 ms / TE 11 ms) slice thickness (ST) of 4 mm; coronal T1 weighted SE (TR 700 ms / TE 11 ms) ST of 4 mm; SPACE sagittalT1 weighted (TR 800 ms / TE 43 ms) ST of 0.7 mm; 2D Intermediate weighted (IW) Fat Saturation (FS) Sagittal (TR 4620 ms / TE 21 ms) ST 3 mm; Coronal 2D IW (TR 4370 ms/ TE 11 ms) ST 3 mm and sagittal T2* 2D (MapIt TR 958 ms, TE 4, 11, 18, 25 y 32 ms) ST 3 mm.
The predefined analysis strategy was by intention to treat. Data were summarized using means and standard deviations (SD), medians and percentiles 25 (p25) and 75 (p75), and counts and percentages. The Shapiro-Wilk test was used to test the normality assumption. For comparisons, we used the unpaired Student´s t test, the Mann-Whitney U test, the paired Student´s t test, and the Wilcoxon matched-pairs signed-ranks test, as appropriate. All tests were two-tailed. A p value < 0.05 was considered statistically significant. All the statistical analyses were performed using Stata 14 (Stata Corp. 2015. Stata Statistical Software: Release 14. College Station, TX: Stata Corp LP).