Trial design
This study is a randomized controlled trial of Evidence Level I from April 2018 to December 2019. The study conforms to the principles of the Declaration of Helsinki. The trial is endorsed by the Shanghai Changzheng Hospital Ethics Committee (CZEC (2016)-02). There is no commercial sponsorship of this study. The trial is registered (ChiCTR1800015379).
Trial participants
All participants learned about research recruitment from outpatient surgeons. To be admitted to the trial, the inclusion criteria for patients were (1) age between 18 and 75 years old, (2) presence of clearly indicated articular cartilage lesions on MRI and Kellgren-Lawrence grade ≤ level 3, (3) obvious knee pain or discomfort lasting for more than 3 months, (4) understanding of the treatment and signed informed consent by the patient or their family, (5) articular cartilage lesions diagnosed by arthroscopy without targeted treatment.
The exclusion criteria for patients were (1) previous surgical procedures for articular cartilage lesions, (2) history of intra-articular injection or history of peri-articular invasive treatments within 3 months of the start of the study, (3) symptoms and imaging findings localized in the patellofemoral joint, (4) presence of malignant neoplasms, (5) active infection anywhere in the body, (6) pregnant or lactating women or women preparing for pregnancy, (7) articular cartilage lesions caused by infectious or gouty arthritis, (8) presence of autoimmune diseases such as rheumatoid arthritis or ankylosing spondylitis, (9) diabetes with fasting blood glucose over 8mmol/ L, (10) general poor health and not recommended for surgery, (11) presence of severe diseases such as cerebral hemorrhage, severe pneumonia, or multiple organ dysfunction, (12) failure to give consent to participate in the study, (13) diagnosis of Charcot joint, (14) findings that may increase patient risk or influence the results of the intervention, (15) other reasons for not being fit for the study.
After being advised about the trial protocol and prior to the start, all patients submitted written informed consent.
Randomization and blinding
Randomization was performed by an independent researcher not involved in the treatment or outcome measurement. After providing informed consent, each eligible patient was randomly assigned a sequence number from a computer-generated random numbers list and randomly separated into one of the two study groups: an experiment group (knee arthroscopic therapy with autologous IPFP cell concentrates) or a control group (knee arthroscopic therapy only). The random number was concealed in sealed an envelope and saved by a researcher who did not participate in the trial. The final unblinding was performed after data collection. Participants were kept blinded to the allocated treatment during the follow-up period. All participants in the experiment group and control group received knee arthroscopic therapy by the same surgeon. Outcome assessors were blinded to the study groups and did not take part in the implementing interventions. During follow-up, all the doctors, radiologists and statisticians were unaware of the study group assignments.
Interventions
Experiment group. All participants in the experiment group received knee arthroscopic therapy by the same surgeon. All the interventions were done in the laminar flow operation room, which was in aseptic condition. After total anesthesia, the surgeon evaluated the medial, lateral, and patellofemoral joint compartments. Then the surgeon performed one or more of the following treatments: debridement; excision of degenerative tears of the menisci, fragments of articular cartilage, chondral flaps, or osteophytes that prevented full extension. Neither abrasion nor microfracture of cartilage lesion was performed. During the knee arthroscopic therapy, partial IPFP tissue was acquired using a standard motorized shaver system. Briefly, 200mL of the mixture containing fat, synovial tissue and 0.9% saline was collected by a sterilized arthroscopic setup of the fat collection system connected with suction apparatus. After being filtered twice using a 30 mesh filter (pore size of 550μm) to remove the synovial tissue, the mixture was centrifuged at 300×g for 5 min [23]. While the liquid supernatant was discarded, 6mL of 0.9% saline was used to mix the lipoaspirate to make cell the concentrates. In total, 5mL of the cell concentrates were injected into the corresponding joint cavity, and another 1mL were was used for identification by flow cytometry [23] and cultured to determine if the cell concentrates were contaminated. (Fig. 1)
The cell concentrates were assayed for cell surface protein expression by flow cytometry (FC500, Beckman Coulter, USA). 100μL from 1mL of freshly isolated cell concentrates were washed twice with phosphate-buffered saline. The antibodies used for the identification of cell concentrates were CD45-FITC, CD44-PE-Cy, CD90-PE, and PE-CD105 (BD Pharmingen, USA). As a negative control, a cell suspension without antibodies was employed following the same procedure. Cell concentrates were incubated with antibodies for 20 minutes at 4°C and then resuspended in fluorescence-activated cell-sorting media and analyzed immediately.
Control group. All participants in the control group received regular knee arthroscopic therapy after anesthesia. During the arthroscopic therapy, same amount of the IPFP tissue was removed under arthroscope, then the joint cavity was treated with routine arthroscopic therapy same as the experiment group. After the arthroscopic therapy, 5mL of 0.9% saline was injected into the treated knee cavity.
Concomitant care and interventions. Patients in both groups were restricted from taking corticosteroids and nonsteroidal anti-inflammatory drugs 1 week before surgery. After the surgery, no corticosteroid was injected into the knee articular cavity. During the rehabilitation period, walking and mild activities were not restricted. Subsequently, the gradual resumption of normal sports or recreational activities was allowed. No analgesics or anti-inflammatory drugs were allowed after the treatment. All post-operational treatment and rehabilitation processes were the same in both groups.
Assessment
All patients were evaluated by an investigator blinded to the group allocation. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) of patients were assessed preoperatively, and at 6-weeks, 12-weeks, 6-months, and 12-months after the intervention to evaluate the functional recovery of the knee joint. The secondary objective of the study was to compare the efficacy of the two methods for pain relief using the Visual Analog Scale (VAS) in both static and motion states at pre-operation and 6-weeks, 12-weeks, 6-months, and 12-months after the intervention. The safety of the two methods was assessed by routine blood analysis, hepatorenal function, and ESR and CRP levels at each follow-up.
Knee magnetic resonance imaging (MRI) was conducted to assess the cartilage regeneration at pre-operation and 12-months after the intervention using Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART). All these evaluations were performed by two well-experienced orthopedists, once disagreements existed, a third one would join in.
Side effects
Adverse reactions were defined as any unexpected events that occurred when the trial was noted and recorded. Serious adverse events (SAEs) were defined as events that were life-threatening or resulted in death, hospitalization, or significant disability.
Statistical analysis
All statistical analyses were performed with SPSS software, version 19.0 (IBM, Armonk, NY). All data were expressed as means and standard deviations. Because the primary outcome was the difference in WOMAC score between baseline and 12 months, sample size was set based on the results of our pilot study and previous study [22] (α risk 0.05, power 0.8, 10% losses to follow-up, changes in WOMAC score 15, and SD 8). The required number of patients should be at least 23. To improve the reliability of our study, we increased the sample size to 30 patients per group.The Student t tests were used to analyze statistical differences between preoperative and follow-up values of WOMAC scores, VAS scores, and MOCART scores. For all the tests, significance was defined as P <0.05.