Inclusion and exclusion criteria
All experimental protocols used in this study were approved by the Ethics Committees of Zhejiang Provincial People’s Hospital. Patients enrolled in this study provided signed written informed consent. Eligible patients included were aged between 18 and 75 years, with OA K-L grades 2 and 3, exhibiting substantial pain and loss of function, failure of conservative therapy, and had an initial pain evaluated at four or greater on a ten-point VAS in the knee joint. On the other hand, exclusion criteria comprised of secondary arthritis (for example, secondary knee osteoarthritis, rheumatoid arthritis, gouty arthritis, and previous articular fractures), contraindicating MRI examination, a history of liposarcoma and other cancers, intra-articular injection of hyaluronic acid or other drugs in the preceding 2 months, end-stage (grade 4) OA, serological positive of Human Immunodeficiency Virus, abdominal hernia, and coagulopathy.
Study design
Procedures related to screening, assessment, fat collection, and SVF injection of patients were performed in private physician examination rooms. Three senior radiologists were responsible for the radiological evaluation and came to a consensus conclusion. The cartilage model was established by professional engineers with the help of a special MRI sequence. During scheduled clinical visits, relevant questionnaires were collected.
To evaluate the grade of OA, an initial X-ray image was used following the K-L criteria, and subsequently, patients belonging to grades 2 and 3 were selected. Afterward, patients who underwent MRI included conventional and three-dimensional fat-suppressed spoiled gradient recalled echo (3D-FS-SPGR) sequences. We used the conventional sequence to record the whole-organ magnetic resonance imaging score (WORMS) and magnetic resonance observation of cartilage repair tissue (MOCART), while the 3D-FS-SPGR sequence was employed to build a 3D cartilage model and measure the related parameters. Using visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire, pain and functional limitation were evaluated. We also examined the range of motion (ROM) during the follow-up period.
Clinical evaluation
Here, the VAS and WOMAC questionnaires were used for the evaluation of pain and functional limitation. In particular, we evaluated the WOMAC score in terms of pain, stiffness, and physical function with a total score ranging from 0 (best health) to 96 (worst health). The maximum score of each subscale was 20, 8, and 68, respectively. The total score of VAS ranged from 0 (best) to 10 (worst). Additionally, we recorded the ROM of the knee joint during the follow-up. Assessments were performed at baseline (before the first injection), 1 month after injection, and during follow-up visits after 3, 6, and 12 months. Finally, we assessed the safety of SVF by analyzing the incidence rate of adverse events (AE) and serious adverse events (SAE).
Radiological assessment
For this clinical study, we performed MRI at baseline, 6, and 12 months. In order to minimize the influence of knee joint loading on the results of MRI, patients were required to rest for 30 minutes before examination. The conventional MRI sequences included T1W1 images in the sagittal plane and PDWI-FS images in the sagittal, coronal, and transverse planes. We employed the MOCART score to examine the cartilage repair, while the WORMS was used for the assessment of the knee [25,26].
Establishment of 3D cartilage model
The MRI scanning was performed on a clinical 3.0T system (GE Healthcare, Waukesha, WI, USA). Using the 3D-FS-SPGR sequence, each patient was examined before SVF injection. Acquisition parameters for the 3D-FS-SPGR sequence were as follows: TE: 3 ms; TR: 14.6 ms; acquisition matrix; 512 x 512; the number of excitations: 2; field-of-view: 15 cm; slice thickness: 1.0 mm; interslice gaps: 0 mm; and receiver BW: ± 41.7 kHz [27].
To build the 3D cartilage model, the original data of the 3D-FS-SPGR sequence was converted to Digital Imaging and Communications in Medicine (DICOM) format and transferred into the Mimics 20.0 software (Materialise, Leuven, Belgium). First, all layers of cartilage defects were detected using 3D-FS-SPGR and conventional sequences. An appropriate segmentation threshold (1849-3445 GV) was set for retaining the healthy cartilage of the knee joint, saving the results as the green mask. Following this, the cartilage defect was segmented by another mask, then saving it as a red mask. The healthy cartilage and cartilage defects of the knee joint segmented by use of different masks are given in Fig. 1. After the layer-by-layer hierarchical image processing, the cartilage model was characterized into six regions, namely, medial femoral condyle (MF), lateral femoral condyle (LF), femoral inter-condylar (T), medial tibia condyle (MT), lateral tibia condyle (LT), and patella (P) [28]. Different color masks represented different areas, while cartilage defects were represented by red masks (Fig. 2a). Then, the cartilage tissue for each layer was preserved, the contours of knee cartilage were calculated, and the cartilage model of each region was established (Fig. 2b). The volume of healthy cartilage, as well as the volume, surface, and thickness of cartilage defects, were measured by the same professional surveyor (Fig. 2c). After one week, the cartilage model was re-established, and the above-mentioned data were measured and averaged.
Fat tissue harvest
For this experiment, patients were not allowed to take aspirin, corticosteroids, and non-steroidal anti-inflammatory drugs within one week before liposuction. Also, they all fasted for liquids and solids at least six hours before the operation. After confirmation of the eligibility criteria, the operator, anesthesiologist, and circulating nurse performed general anesthesia in patients in the supine position. The operation was performed by the same skilled plastic surgeon, who was blinded to patient information. After adequate disinfection of the abdomen and lower extremities, the surgeon made two small incisions of about 5 mm around the umbilical cord and obtained about 100 to 150 ml of adipose tissue from the subcutaneous tissue around the umbilicus using the superwet technique. Thereafter, sutures were used to close the wound, whereas the abdominal binder was used to ensure that the bruises do not occur in the surgical area. Finally, we stored the harvested adipose tissue in a sterile container, followed by packaging it in a portable cryopreservation box. This was done so that the collected adipose tissue would not be damaged on the way to the laboratory.
SVF isolation and injection
Briefly, lipoaspirates were washed with phosphate-buffered saline, while the mesh filter was applied to remove containing residual blood cells and tissue fragments. Next, an equal volume of digestive enzyme (type I collagenase; Worthington, Lakewood, NJ, USA) was mixed with the washed adipose tissue and placed in a shaking incubator at 37 °C for 30 minutes. The resulting mixture was then centrifuged at a rate of 1000 g for 10 minutes and subsequently discarded the supernatant (Eppendorf 5810R, Germany). After this, the remnant SVF at the bottom was resuspended in PBS up to a volume of 4.5-ml SVF, whereas an automatic cell counter (Countstar IC1000, China) was used to quantify cell quantity and viability.
In short, about 4 ml of SVF suspension was injected into the region of the cartilage defect by a trained experienced orthopedic surgeon. Upon the injection of SVF, subcuticular suture and pressure dressing were performed. All the operations were performed by the same experienced orthopedic surgeon.
Statistical analysis
Changes in all follow-up data including VAS, WOMAC, WORMS, and MOCART, among others, were determined using a paired t-test. The discrete data were analyzed by chi-square test. A value of p < 0.05 was considered statistically significant. Data displayed in the graphs are means with standard deviation. All statistical data analysis was executed using SPSS software (version 20.0, IBM Corporation, NY, US).