Outcome measures
anaphylaxis, fever, swelling, injection-site reaction, infections, knee osteoarthritis, pain, SAEs, TMA, Knee osteoarthritis, chills, joint stiffness
ANAPHYLAXIS, SUDDEN DEATH, OSTEOARTHRITIS, ALLERGIC REACTION, ADVERSE EVENTS, NEOPLASMS, RENAL AND URINARY DISORDERS, SKIN AND SUBCUTANEOUS TISSUE DISORDER, INFECTIONS, KNEE OSTEOARTHRITIS, KNEE OSTEOARTHRITIS, GASTROINTESTINAL DISORDERS, NEUROLOGICAL DISORDERS
Safety assessment, as primary outcome, was conducted by recording any severe adverse events (SAEs) and adverse events (AEs) during the follow-up. AEs and SAEs including allergic reaction, anaphylaxis, fever, chills, pain, injection-site reaction, sudden death, not otherwise specified (NOS), joint swelling, joint stiffness, neurological disorders, skin and subcutaneous tissue disorders; neoplasms benign, malignant, and unspecified gastrointestinal disorders; and infections, renal and urinary disorders were evaluated according to common terminology criteria for adverse events (CTCAE) version 4.0.Efficacy of AD-MSC’s injection was evaluated and comprised by several questionnaires including Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), VAS, and knee osteoarthritis outcome score (KOOS), and Short Form 36 (SF-36) questionnaires at the baseline and in 3, 6, and 12 months after injection. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), KOOS, VAS, and SF-36 are self-administered questionnaires which completed by patients. The WOMAC consists of 24 items that are divided into three subscales: pain (5 items), stiffness (2 items), and physical function (17 items). Each item was scored on a Likert scale ranging from 0 to 4, with higher scores indicating more severe symptoms. To calculate the total WOMAC score, the raw scores were summed (0–96) and then transformed to a scale of 0–100. Knee osteoarthritis outcome score (KOOS) consists of 42 items, divided into five subscales: pain, symptoms, daily function, sport and recreation, and quality of life. Each item was scored on a Likert scale ranging from 0 to 4, where 0 represents no difficulty or symptom, and 4 represents extreme difficulty or severe symptom. The KOOS score was calculated by summing the scores for each subscale and averaging them, which higher scores indicate better knee function and less severe OA symptoms [Inflammatory changes following the injection have been assessed by measuring the levels of some specific and inflammatory biomarkers including cartilage oligomeric matrix protein (COMP), hyaluronic acid (HA), matrix metalloproteinase-3 (MMP-3), IL-6, and IL-10 in blood serum at baseline, 1 week, and 3 months after injection. The levels of these biomarkers have been measured by the enzyme-linked immunosorbent assay (ELISA). To measure serum COMP, HA, Il-10, and IL-6 levels using ELISA, human COMP ELISA kit (Cat. No: E1486Hu), human HA Elisa kit (Cat. No: E1374Hu), human IL-10 ELISA kit (Cat. No: E0102Hu), and human IL-6 ELISA kit (Cat. No: DE4640) were used. We followed the manufacturer’s instruction for each kit.Another monitoring strategy used in this study was evaluation of the cell surface CD markers. Cells obtained from peripheral blood samples were analyzed by flow cytometry for the expression of CD3, CD4, CD8, and CD25 markers. Analysis of cell markers was performed at baseline, and 1, 4, 12, and 24 weeks after injection. In this regard, the samples were processed to obtain a single-cell suspension and remove any debris or aggregates that may interfere with the analysis by adding PBS. The next step was to stain the cells with fluorescent-conjugated antibodies that recognize specific cell surface markers. Different CD markers were detected with their specific antibodies which conjugated with fluorescent dyes to allow their simultaneous detection. After staining, the cells were washed to remove unbound antibodies. Data collection and analysis of the fluorescent intensities were made using a flow cytometer (Becton Dickinson, New Jersey, USA) at Royan institute.Magnetic resonance imaging (MRI) assessment was conducted at baseline and 12 months after the injection. Fast spoiled gradient-echo (FSPGR) MRI protocol was used in sagittal plane using 3 Tesla system (Magnetom Tim Trio; Siemens, Erlangen, Germany). The thickness of distal femoral cartilage and proximal tibial cartilage was evaluated in femur lateral central (FLC), femur lateral posterior (FLP), femur lateral anterior (FLA), femur medial central (FMC), femur medial posterior (FMP), femur medial anterior (FMA), tibia lateral anterior (TLA), tibia lateral central (TLC), tibia lateral posterior (TLP), tibia medial anterior (TMA), tibia medial central (TMC), and tibia medial posterior (TMP). The one-way repeated measures analysis of variance (ANOVA) has been used to evaluate the validity of cartilage thickness changes at baseline and 1-year post-injection follow-up.