Abstract
The aim of the article is to compare the clinical and radiological outcomes between single and double stromal vascular fraction (SVF) cell injections in patients with knee osteoarthritis (OA). We included 54 patients treated for varus knee OA with intra-articular SVF cell injection. They were divided into two groups: those who received one injection and those who received two. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, knee range of motion, and knee muscle force were assessed at baseline and 3, 6, 12, and 24 months after the first injection. The preoperative hip-knee-ankle (HKA) angle was evaluated using plain radiographs, and T2 mapping values were assessed. The total WOMAC score improved significantly in the single injection group from 3 to 24 months, but the total WOMAC score in the double injection group improved significantly at 24 months. The T2 mapping values in both the groups improved, with a significant difference at 12 months. The preoperative mean HKA angle and the correlation coefficients between the HKA angle and the total WOMAC score and between the HKA angle and the T2 mapping value of the medial femur were significant. In conclusion, double injections may provide more satisfactory treatment outcomes in patients with severe varus knee alignment. This clinical trial is registered in the Japanese Ministry of Health, Labour and Welfare (URL: https://saiseiiryo.mhlw.go.jp/published_plan/index/2) with the registration name “Cell transplantation therapy for osteoarthritis using autologous subcutaneous adipose tissue-derived regenerative (stem) cells (ADRCs),” and the registration number was “PB5160012.”
Keywords: stromal vascular fraction, regenerative therapy, knee osteoarthritis
Introduction
Osteoarthritis (OA) of the knee results from chronic cartilage degeneration and is the most common musculoskeletal disorder that causes pain, stiffness, and decreased function in daily activities1,2. Recently, the authors reported promising results of direct intra-articular injection of marrow-derived stem cells (MSCs) and adipose-derived stem cells (ADSCs) into the knee joint for the treatment of knee OA3,4, but the preparation of MSCs and ADSCs involves the isolation and culture of the cells and different procedures 5 .
Adipose-derived stromal vascular fraction (SVF) cells are composed of heterogeneous cells, including ADSCs, macrophages, pericytes, fibroblasts, blood cells, and vessel-forming cells, obtained via mechanical or enzymatic digestion of adipose tissue without culture or multiple passages5,6. The SVF cell collection method is simpler than that of other stem cell sources. Its use allows for a single-step surgical procedure for regenerative therapy 7 . Previous studies have suggested that the safety and efficacy of SVF cells are equal to those of other stem cells in several settings, such as nerve regeneration, hypertrophic scar remodeling, treatment of acute myocardial infarction, and cartilage regeneration in animal models8–12. Thus, SVF cells are a promising source of regenerative therapy. A recent study reported that intra-articular SVF cell injection into the knee joint had excellent clinical results and cartilage repair in patients with knee OA 13 . However, a detailed evaluation of SVF cell treatment for knee OA is insufficient. Some patients were unsatisfied by only one injection, and the second injection was required in some cases for satisfactory treatment. The multiple injections of traditional intra-articular injections, such as hyaluronic acid (HA), provided higher benefits than a single injection for pain improvement in patients with knee OA 14 . However, an investigation on the benefits of two injections of SVF cells for knee OA has not yet been reported.
Therefore, this retrospective study aimed to compare the clinical and radiological outcomes between single and double SVF injections for patients with knee OA and investigate the negative factors for the treatment of knee OA using intra-articular SVF cell injection.
Methods
Study Design and Patients
This retrospective study was approved by the Review Board for Human Research of Sobajima Clinic (reference number: SC002-1M and SC002-2M) and the Kobe University Graduate School of Medicine (reference number: 170181). All methods in the present study were carried out in accordance with relevant guidelines and regulations. All the patients provided informed consent for inclusion in the study. Our inclusion criteria were patients with grade I to IV varus knee OA evaluated using the Kellgren–Lawrence (KL) classification, exhibiting substantial pain and difficulty in performing daily activities despite conservative treatments, including rehabilitation, medication, and intra-articular injections of HA or steroids for ≥3 months. Patients with a history of knee joint trauma, severe bony defects, and an active or previous joint infection were excluded. In addition, patients were excluded if they did not have adequate clinical and radiographic data for analysis. After an intra-articular injection of SVF cells, the patients were informed to regularly visit the hospital for rehabilitation by a physical therapist, in addition to performing daily home exercises by themselves according to a standardized rehabilitation protocol of the hospital. One year after the first SVF injection, patients with more than 16.5 points of the total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were judged to have not achieved treatment success 15 , and the second injection was recommended. These patients were treated with a second injection of SVF. Finally, 54 patients (54 knees; 14 men and 40 women; mean age: 68.9 ± 9.9 years) who met the inclusion and exclusion criteria were selected for this retrospective analysis. They were divided into two groups: those who had only one injection (single injection group; 30 knees; 8 men and 22 women; mean age: 68.8 ± 8.2 years) and those who had a second injection (double injection group; 24 knees; 6 men and 18 women; mean age: 69.1 ± 11.8 years).
SVF Preparation
SVF cells were prepared from the abdominal or breech subcutaneous fat by liposuction using the Celution® 800/CRS System (Cytori Therapeutics Inc., San Diego, CA, USA). Briefly, all the subjects underwent a liposuction procedure under general anesthesia to obtain 100–360 ml of adipose tissue. The subcutaneous adipose tissue was removed, minced, and incubated with Celase® GMP (Worthington Biochemical Corp., San Diego, CA, USA), which is a mixture of highly purified collagenase, to digest the aspirated adipose tissue. After digestion, the SVF cells were concentrated by centrifugation, washed to remove the Celase® reagent, and extracted from the system. The viable cell count of SVF cells was determined using the NC-100™ NucleoCounter® Automated Cell Counting System (Chemometec, Allerod, Denmark).
Intra-articular Injection
The re-suspended SVF cells in 5 ml of phosphate-buffered saline (PBS) were injected into the knee joint using a superolateral patella approach under ultrasound guidance without anesthesia. We administered 2.5 × 107 SVF cells in the first injection to each patient, according to previously reported guidelines 16 , and 1.4 × 107 in the second injection for the double injection group. The mean cell viability of the SVF cells was 90.0% ± 4.2% in the first injection and 76.7% ± 4.7% in the second injection of the double injection group.
Freezing and Storage of SVF Cells
In all the cases, the remaining cells were cryopreserved using a previously described procedure 17 . Briefly, cells were frozen in 20% human serum albumin (10 g/50 ml; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) and 10% dimethyl sulfoxide (DMSO) in lactated Ringer’s solution at −80°C, and then cooled at −1°C/min from 4°C to −50°C, and at −10°C/min to −80°C. In the cases that required two injections, the cryopreserved SVF cells were thawed in a water bath at 37°C for 2 min, washed rapidly, and suspended in 10 volumes of PBS. The cells were then centrifuged at 1,500 rpm for 6 min, washed in PBS, and suspended in 100 μl of PBS.
Clinical Evaluations
Clinical evaluations, including WOMAC score, knee range of motion (ROM), and muscle force of the knee, were performed at baseline and 3, 6, 12, and 24 months following the first intra-articular injection of SVF cells. The WOMAC scores were linearly transformed to a 0–100 scale, with higher scores indicating more severe impairment. Clinical evaluations were performed by an experienced physiotherapist. To measure the muscle force of knee extension and flexion, a hand-held dynamometer was used 18 . Briefly, the patient was placed in a prone position with the knee flexed at 90°, and a sensor was placed at the center of the lower leg. The patients were asked to bend and straighten their knees for 3 s against manual resistance. The measurements were taken three times, and the average value was recorded.
Radiologic Evaluations
The hip-knee-ankle (HKA) angle was measured on a full-length weight-bearing radiograph in the standing position before the first SVF cell injection. The HKA angle was expressed as the deviational angle from 180°, with positive values for varus malalignment. T2 mapping values were measured using a 1.5-T magnetic resonance imaging (MRI) unit (Signa Excite HDx; GE Healthcare, Waukesha, WI, USA). We measured T2 mapping values at baseline and 12 and 24 months after the first SVF cell injection according to a previously reported method 13 . Briefly, we selected a central slice that passed through the center of the weight-bearing cartilage surrounded by the anterior and posterior margins of the meniscus on a sagittal slice and two slices neighboring the central slice anteriorly and posteriorly. The region of interest (ROI) was then set at the full-thickness cartilage of the medial and lateral sides of the femoral condyle and the tibial plateau on the central, anterior, and posterior slices of the coronal image (Fig. 1). Finally, the T2 mapping values of 12 ROIs were measured by an independent orthopedic surgeon with 15 years of experience in MRI analysis.
Figure 1.
Method of calculating T2 mapping value of cartilage. (A) In a sagittal T1-weighted MR image, a central slice (yellow dotted line) passing the center between the anterior and posterior margins of the meniscus was selected. (B) In a coronal T2-weighted MR image on the central slice, the region of interest (ROI) was set at the cartilage (red line) of the medial and lateral femoral condyle and medial and lateral tibial plateau. (C) In a coronal T2 map MR image, the T2 mapping value corresponding to placement positions of ROI was calculated. MR: magnetic resonance.
Safety Evaluation
The safety was assessed based on the vital signs, physical examination, and adverse events, and all outcomes were recorded.
Statistical Analyses
All the values were expressed as mean ± standard deviations. Data analyses were performed using IBM SPSS statistical software (version 21; IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was used to analyze normally distributed data. Repeated measures analysis of variance was used to detect the mean differences in the independent variables of time for the dependent variables of the WOMAC score and T2 mapping values of ROIs. The unpaired t test was performed to compare parameters between the two groups (single and double injection groups). Differences in sex and KL classification between the two groups were also analyzed using the chi-square test. Pearson’s correlation coefficient was used to investigate the correlation between HKA angles and clinical and imaging evaluations. Statistical significance was set at P < 0.05. A statistical power analysis was performed prior to the study using G*Power 3 19 , and a power analysis indicated that 21 patients were required in each group when using a type I error (a) of 0.05, power (1 − b) of 0.8. In addition, the minimum sample size required to achieve a modest correlation between HKA angles and clinical and imaging evaluations was 29 patients, when alpha was 0.05 and power was 0.8.
Results
Patient Population
As summarized in Table 1, the two groups were well matched in terms of sex, age, and body mass index. There were no significant differences in preoperative OA progression based on KL classification.
Table 1.
Patient Characteristics.
| Characteristics | Single injection group (n = 30) | Double injection group (n = 24) | P value |
|---|---|---|---|
| Sex (male/female) | 8/22 | 6/18 | n.s. |
| Age | 68.8 ± 8.2 | 69.1 ± 11.8 | n.s. |
| Height, cm | 156.9 ± 7.6 | 156.8 ± 9.8 | n.s. |
| Weight, kg | 62.7 ± 8.9 | 62.3 ± 12.4 | n.s. |
| Body mass index, kg/m2 | 25.4 ± 2.5 | 25.2 ± 3.8 | n.s. |
| Kellgren–Lawrence classification (%) | n.s. | ||
| I | 0 (0) | 0 (0) | |
| II | 4 (13) | 2 (8) | |
| III | 16 (53) | 12 (50) | |
| IV | 10 (33) | 10 (42) |
Clinical Outcomes
Table 2 presents the clinical evaluations of the groups. In the single injection group, the total WOMAC score was significantly improved at 3, 6, 12, and 24 months compared with that at baseline. In the double injection group, the clinical scores improved at 3, 6, and 12 months, but the difference was not significant; however, the scores significantly improved at 24 months. In addition, there were significant differences in the total WOMAC scores between the two groups at 3, 6, and 12 months. The WOMAC pain, stiffness, and function scores showed similar results. The mean extension angle of the knee joint significantly improved from baseline to 24 months. The flexion angle of the knee and the muscle force of knee extension and flexion were improved in both groups, but there was no significant difference from baseline. There was no statistical difference in ROM and muscle forces between the two groups.
Table 2.
Clinical Evaluation Results.
| Western Ontario and McMaster Universities Osteoarthritis Index | ||||
|---|---|---|---|---|
| Total score | All cases | Single injection group | Double injection group | P value |
| Preoperative | 38.8 ± 17.0 | 35.4 ± 18.3 | 43.0 ± 14.6 | n.s. |
| 3 months | 28.4 ± 14.7 † | 23.1 ± 13.2 † | 34.9 ± 14.0 | 0.003* |
| 6 months | 26.2 ± 16.2 † | 21.1 ± 14.6 † | 32.5 ± 16.2 | 0.010* |
| 12 months | 27.0 ± 17.6 † | 21.8 ± 16.6 † | 33.6 ± 17.1 | 0.014* |
| 24 months | 25.4 ± 17.1 † | 23.3 ± 18.2 † | 28.1 ± 15.7 † | n.s. |
| Pain | All cases | Single injection group | Double injection group | P value |
| Preoperative | 65.2 ± 26.1 | 59.0 ± 25.8 | 72.9 ± 25.0 | n.s. |
| 3 months | 45.6 ± 26.4 † | 37.7 ± 25.8 † | 55.4 ± 24.4 | 0.013* |
| 6 months | 43.6 ± 26.2 † | 37.2 ± 25.9 † | 51.7 ± 24.7 † | 0.042* |
| 12 months | 43.9 ± 27.9 † | 36.8 ± 29.1 † | 52.7 ± 24.2 † | 0.037* |
| 24 months | 42.6 ± 23.8 † | 39.5 ± 26.7 † | 46.5 ± 19.3 † | n.s. |
| Stiffness | All cases | Single injection group | Double injection group | P value |
| Preoperative | 44.4 ± 21.7 | 42.5 ± 20.9 | 46.9 ± 22.8 | n.s. |
| 3 months | 34.3 ± 18.8 | 27.9 ± 16.3 † | 42.2 ± 19.1 | 0.005* |
| 6 months | 31.9 ± 23.4 † | 26.7 ± 21.2 † | 38.5 ± 24.7 | n.s. |
| 12 months | 32.2 ± 26.7 † | 27.9 ± 21.7 † | 37.5 ± 20.9 | n.s. |
| 24 months | 31.0 ± 21.5 † | 27.5 ± 19.8 † | 35.4 ± 23.2 | n.s. |
| Function | All cases | Single injection group | Double injection group | P value |
| Preoperative | 30.3 ± 17.0 | 27.6 ± 19.2 | 33.7 ± 13.4 | n.s. |
| 3 months | 22.6 ± 13.1 | 18.2 ± 11.8 | 28.1 ± 12.8 | 0.005* |
| 6 months | 20.4 ± 14.9 † | 15.8 ± 12.8 † | 26.1 ± 15.5 | 0.010* |
| 12 months | 21.5 ± 16.5 † | 16.7 ± 14.8 | 27.5 ± 16.8 | 0.015* |
| 24 months | 19.7 ± 16.5 † | 18.0 ± 17.4 | 21.8 ± 15.2 † | n.s. |
| Range of motion of the knee (°) | ||||
| Extension | All cases | Single injection group | Double injection group | P value |
| Preoperative | −6.1 ± 5.4 | −5.2 ± 5.2 | −7.3 ± 5.5 | n.s. |
| 3 months | −5.0 ± 5.2 | −4.8 ± 5.8 | −5.2 ± 4.5 | n.s. |
| 6 months | −4.6 ± 4.6 | −4.2 ± 4.7 | −5.2 ± 4.5 | n.s. |
| 12 months | −3.9 ± 4.2 | −3.7 ± 4.3 | −4.2 ± 4.1 | n.s. |
| 24 months | −3.9 ± 4.0 † | −3.2 ± 3.8 | −4.8 ± 4.0 | n.s. |
| Flexion | All cases | Single injection group | Double injection group | P value |
| Preoperative | 128.7 ± 16.5 | 129.2 ± 16.2 | 128.1 ± 17.2 | n.s. |
| 3 months | 133.3 ± 14.3 | 133.5 ± 14.6 | 133.1 ± 14.3 | n.s. |
| 6 months | 132.1 ± 15.5 | 133.2 ± 14.5 | 130.8 ± 16.9 | n.s. |
| 12 months | 130.0 ± 15.9 | 131.3 ± 14.1 | 128.3 ± 18.1 | n.s. |
| 24 months | 131.1 ± 14.9 | 131.3 ± 13.2 | 130.8 ± 17.0 | n.s. |
| Muscle force (N m) | ||||
| Extension (quadriceps) | All cases | Single injection group | Double injection group | P value |
| Preoperative | 206.6 ± 79.6 | 201.3 ± 75.8 | 213.2 ± 85.2 | n.s. |
| 3 months | 222.8 ± 84.0 | 229.4 ± 87.4 | 214.7 ± 80.7 | n.s. |
| 6 months | 228.7 ± 78.2 | 227.6 ± 81.9 | 230.0 ± 75.0 | n.s. |
| 12 months | 238.8 ± 84.0 | 253.5 ± 92.3 | 220.4 ± 70.0 | n.s. |
| 24 months | 217.3 ± 86.9 | 217.0 ± 93.8 | 217.7 ± 76.9 | n.s. |
| Flexion (Hamstrings) |
All cases | Single injection group | Double injection group | P value |
| Preoperative | 102.9 ± 36.2 | 104.9 ± 37.1 | 100.4 ± 35.6 | n.s. |
| 3 months | 107.8 ± 31.2 | 113.9 ± 31.5 | 100.1 ± 29.6 | n.s. |
| 6 months | 114.1 ± 31.0 | 121.8 ± 35.4 | 104.6 ± 21.5 | n.s. |
| 12 months | 118.1 ± 32.0 | 122.4 ± 32.8 | 112.7 ± 30.8 | n.s. |
| 24 months | 109.7 ± 27.0 | 114.3 ± 26.9 | 106.4 ± 27.3 | n.s. |
Statistically significant between the single injection group and the double injection group; †statistically significant compared with preoperative outcome.
Imaging Evaluation
Table 3 summarizes the imaging evaluations of both groups. The mean preoperative HKA angle was significantly different between the two groups: 185.9° in the single injection group and 188.1° in the double injection group. The mean T2 mapping values of all areas (the medial femur, medial tibia, lateral femur, and lateral tibia) in the anterior slice were significantly improved in both groups. In the center and posterior slices, the mean T2 mapping values of the medial tibia and lateral femur were significantly improved in the single injection group. In addition, the T2 mapping values of the medial femur in the anterior and central slices were significantly lower in the single injection group than in the double injection group.
Table 3.
Imaging Evaluation Results.
| Hip-knee-ankle angle | All cases | Single injection group | Double injection group | P value |
|---|---|---|---|---|
| Preoperative | 186.9 ± 3.6 | 186.1 ± 3.2 | 188.0 ± 3.8 | 0.047* |
| Anterior T2 mapping value | ||||
| Medial femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 52.6 ± 7.3 | 51.9 ± 6.2 | 53.4 ± 8.6 | n.s. |
| 12 months | 51.0 ± 5.7 | 49.4 ± 4.2 | 53.0 ± 6.8 | 0.020* |
| 24 months | 48.6 ± 4.7 † | 48.9 ± 4.1 † | 48.4 ± 5.4 † | n.s. |
| Medial tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 43.8 ± 7.2 | 43.8 ± 7.8 | 43.9 ± 6.5 | n.s. |
| 12 months | 40.5 ± 4.9 † | 40.3 ± 5.0 | 40.8 ± 4.8 | n.s. |
| 24 months | 39.8 ± 4.9 † | 39.8 ± 5.3 † | 39.8 ± 4.5 † | n.s. |
| Lateral femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 44.2 ± 4.8 | 44.3 ± 5.7 | 44.2 ± 3.5 | n.s. |
| 12 months | 42.2 ± 4.2 | 41.7 ± 3.6 | 42.9 ± 4.9 | n.s. |
| 24 months | 41.2 ± 4.9 † | 40.8 ± 4.9 † | 41.1 ± 4.1 † | n.s. |
| Lateral tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 39.9 ± 3.8 | 40.1 ± 4.1 | 39.7 ± 3.3 | n.s. |
| 12 months | 37.8 ± 3.8 † | 37.8 ± 4.0 | 37.7 ± 3.5 | n.s. |
| 24 months | 37.3 ± 4.0 † | 37.5 ± 4.3 † | 37.1 ± 3.6 † | n.s. |
| Central T2 mapping value | ||||
| Medial femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 52.1 ± 8.2 | 50.4 ± 7.1 | 54.3 ± 9.0 | n.s. |
| 12 months | 49.8 ± 6.5 | 48.2 ± 5.3 | 51.9 ± 7.4 | 0.045* |
| 24 months | 49.3 ± 6.3 | 48.3 ± 5.1 | 50.6 ± 7.6 | n.s. |
| Medial tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 42.3 ± 4.4 | 42.1 ± 4.0 | 42.6 ± 5.0 | n.s. |
| 12 months | 39.9 ± 5.6 † | 38.7 ± 3.8 † | 41.5 ± 7.1 | n.s. |
| 24 months | 38.8 ± 5.2 † | 38.5 ± 5.5 † | 39.2 ± 5.0 | n.s. |
| Lateral femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 47.5 ± 5.0 | 47.8 ± 4.8 | 47.1 ± 5.2 | n.s. |
| 12 months | 46.6 ± 4.2 | 46.3 ± 3.3 | 46.9 ± 5.2 | n.s. |
| 24 months | 45.0 ± 6.2 † | 44.6 ± 6.1 † | 45.6 ± 6.5 | n.s. |
| Lateral tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 38.2 ± 3.7 | 38.1 ± 4.1 | 38.5 ± 3.4 | n.s. |
| 12 months | 37.9 ± 4.9 | 38.4 ± 5.9 | 37.3 ± 3.2 | n.s. |
| 24 months | 37.3 ± 5.5 † | 37.9 ± 6.1 | 36.6 ± 4.6 | n.s. |
| Posterior T2 mapping value | ||||
| Medial femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 51.9 ± 6.7 | 51.4 ± 7.8 | 52.4 ± 5.3 | n.s. |
| 12 months | 51.1 ± 6.4 | 49.7 ± 4.4 | 52.9 ± 8.0 | n.s. |
| 24 months | 50.3 ± 6.2 | 49.4 ± 5.6 | 51.3 ± 6.9 | n.s. |
| Medial tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 41.5 ± 4.2 | 40.9 ± 3.4 | 42.2 ± 5.1 | n.s. |
| 12 months | 40.3 ± 4.7 | 40.0 ± 4.4 | 40.7 ± 5.2 | n.s. |
| 24 months | 38.9 ± 4.6 † | 38.3 ± 4.5 † | 39.6 ± 4.8 | n.s. |
| Lateral femur | All cases | Single injection group | Double injection group | P value |
| Preoperative | 47.6 ± 4.9 | 47.6 ± 4.4 | 47.6 ± 5.6 | n.s. |
| 12 months | 45.1 ± 5.2 † | 44.7 ± 4.4 | 45.5 ± 6.1 | n.s. |
| 24 months | 44.0 ± 5.2 † | 44.2 ± 5.3 † | 43.9 ± 5.1 | n.s. |
| Lateral tibia | All cases | Single injection group | Double injection group | P value |
| Preoperative | 38.5 ± 4.5 | 38.4 ± 5.0 | 38.6 ± 3.9 | n.s. |
| 12 months | 37.2 ± 4.2 | 37.4 ± 4.7 | 36.9 ± 3.4 | n.s. |
| 24 months | 37.3 ± 5.3 | 37.3 ± 5.2 | 37.3 ± 5.6 | n.s. |
Statistically significant between the single injection group and the double injection group; †statistically significant compared with preoperative outcome.
Correlation Coefficient With HKA Angle
There were significant positive correlations between the preoperative HKA angle and total WOMAC score at 6 and 12 months, stiffness score at 12 months, and function score at 6 and 12 months (Table 4). In addition, there were significant positive correlations between the preoperative HKA angle and T2 mapping values of the medial femur in the anterior and central slices at 12 months postoperatively (Table 5).
Table 4.
Correlation Coefficients Between WOMAC Score and Preoperative Hip-Knee-Ankle Angle.
| Total Western Ontario and McMaster Universities Osteoarthritis Index | Hip-knee-ankle angle | |
|---|---|---|
| Total score | r | P value |
| Preoperative | 0.140 | n.s. |
| 3 months | 0.119 | n.s. |
| 6 months | 0.271 | 0.048* |
| 12 months | 0.280 | 0.042* |
| 24 months | 0.247 | n.s. |
| Pain | r | P value |
| Preoperative | 0.114 | n.s. |
| 3 months | 0.164 | n.s. |
| 6 months | 0.218 | n.s. |
| 12 months | 0.197 | n.s. |
| 24 months | 0.161 | n.s. |
| Stiffness | r | P value |
| Preoperative | −0.001 | n.s. |
| 3 months | 0.118 | n.s. |
| 6 months | 0.222 | n.s. |
| 12 months | 0.219 | n.s. |
| 24 months | 0.319 | 0.020* |
| Function | r | P value |
| Preoperative | 0.117 | n.s. |
| 3 months | 0.110 | n.s. |
| 6 months | 0.273 | 0.047* |
| 12 months | 0.303 | 0.027* |
| 24 months | 0.252 | n.s. |
WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index.
Statistically significant between the single injection group and the double injection group.
Table 5.
Correlation Coefficients Between T2 Mapping Values and Preoperative Hip-Knee-Ankle Angle.
| Medial femur (anterior slice) | Hip-knee-ankle angle | |
|---|---|---|
| r | P value | |
| Preoperative | 0.114 | n.s. |
| 12 months | 0.355 | 0.010* |
| 24 months | 0.200 | n.s. |
| Medial tibia (anterior slice) | r | P value |
| Preoperative | 0.190 | n.s. |
| 12 months | 0.109 | n.s. |
| 24 months | 0.157 | n.s. |
| Lateral femur (anterior slice) | r | P value |
| Preoperative | −0.203 | n.s. |
| 12 months | −0.062 | n.s. |
| 24 months | −0.004 | n.s. |
| Lateral tibia (anterior slice) | r | P value |
| Preoperative | 0.001 | n.s. |
| 12 months | 0.027 | n.s. |
| 24 months | −0.170 | n.s. |
| Medial femur (center slice) | r | P value |
| Preoperative | 0.288 | 0.036* |
| 12 months | 0.381 | 0.005* |
| 24 months | 0.220 | n.s. |
| Medial tibia (center slice) | r | P value |
| Preoperative | 0.167 | n.s. |
| 12 months | 0.224 | n.s. |
| 24 months | 0.326 | 0.018* |
| Lateral femur (center slice) | r | P value |
| Preoperative | 0.005 | n.s. |
| 12 months | −0.038 | n.s. |
| 24 months | −0.023 | n.s. |
| Lateral tibia (center slice) | r | P value |
| Preoperative | 0.007 | n.s. |
| 12 months | −0.077 | n.s. |
| 24 months | −0.028 | n.s. |
| Medial femur (posterior slice) | r | P value |
| Preoperative | 0.257 | n.s. |
| 12 months | 0.249 | n.s. |
| 24 months | 0.303 | 0.027* |
| Medial tibia (posterior slice) | r | P value |
| Preoperative | 0.107 | n.s. |
| 12 months | 0.239 | n.s. |
| 24 months | 0.204 | n.s. |
| Lateral femur (posterior slice) | r | P value |
| Preoperative | −0.249 | n.s. |
| 12 months | 0.016 | n.s. |
| 24 months | 0.048 | n.s. |
| Lateral tibia (posterior slice) | r | P value |
| Preoperative | −0.126 | n.s. |
| 12 months | 0.094 | n.s. |
| 24 months | −0.020 | n.s. |
Statistically significant correlations between T2 mapping values and preoperative hip-knee-ankle angle.
Safety Evaluation
All the intra-articular SVF cell injections into the knee joint did not cause any death or life-threatening complications in any patient. Furthermore, there were no moderate adverse events, such as infection, throughout the follow-up periods in the two groups. However, mild adverse events, such as swelling and pain of the knee, were observed in 9.3% and 8.3% of the patients after the first and second injections, respectively. The symptoms resolved within 3 days in all cases.
Discussion
The most important finding of the present study was that even a single injection of SVF cells into the knee joints resulted in significant clinical effects in patients with severe varus knee OA, though not satisfactory, and two SVF cell injections may contribute to the satisfactory treatment in these patients. To the best of our knowledge, this is the first study to compare the clinical and imaging evaluations between a single injection and double injections of SVF cells into the knee joint for the treatment of knee OA. Based on this finding, intra-articular SVF cell injection into the knee joint may be determined as a safe and excellent treatment for knee OA, but two injections may be required for sufficient clinical effects in patients with severe varus knee OA.
In this study, both clinical outcomes and imaging evaluation were improved following the intra-articular SVF injection. Some studies suggested the therapeutic effects of SVF on the OA joint12,20. A previous author reported that SVF secreted high levels of prochondrogenic cytokines including transforming growth factor (TGF)-β and interleukin (IL)-10, and TGF-β could contribute to the regeneration of chondrocytes via the Smad2/3 signaling pathway 21 . IL-10 also could contribute to the therapeutic effects on OA joint through suppression the joint inflammatory 22 . Our results were in line with the previous studies.
A previous study investigated the cutoff score that reflects the treatment success 1 year after the treatment of knee OA using total knee replacement, and they suggested a total WOMAC score threshold of 16.5 15 . Thus, we recommended patients with a total WOMAC score of greater than 16.5 points for the second SVF cell injection in our institution, and patients were divided into a single injection group and a double injection group. In the present study, the mean total WOMAC score improved after the first injection; however, there was a significant difference between the two groups at 12 months after the first injection. In the double injection group, there was an additional improvement in the WOMAC score following the second injection, and the significant difference between the two groups disappeared. This result suggests that some patients did not achieve sufficient improvement with only one injection, and two injections contributed to the satisfactory treatment. The potential of two injections of stem cells for regenerative therapy has been previously reported in animal models 23 . In addition, recent studies reported that multiple intra-articular injections of MSCs and ADSCs contributed to the improvement of the cartilage defect in knee OA24,25; however, to the best of our knowledge, there are no reports on injections of SVF cells for the treatment of knee OA. The present study is the first report of two injections of SVF cells for regenerative therapy of knee OA. Cryopreserved SVF cells were used for the second injection in this study. A previous study suggested that the viability of cryopreserved SVF cells is almost 70% 26 , and the viability of the cryopreserved SVF cells used in our study is in line with this. A previous study reported that the therapeutic potential of cryopreserved SVF cells was almost equal to that of fresh SVF cells for bone healing in an animal model 27 . Thus, the present study suggests that cryopreserved SVF cells may be helpful for two injections for the treatment of knee OA.
Our results showed that the patients in the double injection group had more severe varus malalignment of the knee joint in a weight-bearing position than those in the single injection group. In addition, there were significant correlations between the HKA angle and WOMAC scores and the T2 mapping values of the medial femur at 12 months after the first injection. Previous studies reported that a severe varus knee alignment may increase knee pain, upregulate inflammatory cytokines in serum, and be associated with knee OA progression28,29. Furthermore, Otsuki et al. 30 suggested that the increased varus knee angle correlated with higher OA grade with KL classification, and the HA and chondroitin sulfate content of knee cartilage in the medial femoral condyle were significantly decreased in the severe varus knee joint with a femorotibial angle ≥190°. T2 mapping is a recently validated method of cartilage quality assessment using MRI that facilitates the detection of changes in water and collagen content. T2 mapping reflects the degree of articular cartilage degeneration31,32. Thus, our clinical and imaging evaluations in the present study support previous studies.
This study has some limitations. First, no control group was included in this study. A larger-scale study with appropriate control is required for further application. Second, clinical and imaging evaluations were only performed in the short term; thus, further investigations with longer follow-up periods are required. Finally, the clinical evaluation in this study was performed preoperatively and at 3, 6, 12, and 24 months after the first injection. We did not evaluate sufficient clinical data after the second injection; therefore, a more detailed investigation of multiple injections should be performed in the future.
Conclusion
The detailed evaluation of the present study showed that the intra-articular SVF cell injection into the knee joint for the treatment of knee OA resulted in the improvement of WOMAC score and T2 mapping values of the knee joint. However, two injections may be required for the satisfactory treatment of patients with severe varus knee alignment.
Acknowledgments
I would like to express my deepest gratitude to the following individuals who have contributed to the completion of this article.
Footnotes
Authors’ Contributions: MF analyzed the data, and wrote the paper. TM designed the study and revised the paper. SS and HI performed the experimental study. MT supported the analysis of data. TM and RK supervised the study. All authors read and approved the final manuscript.
Data Availability Statement: The data are available upon request from the corresponding author.
Ethical Approval: The present study was approved by the Review Board for Human Research of Sobajima Clinic (reference number: SC002-1M and SC002-2M) and the Kobe University Graduate School of Medicine (reference number: 170181).
Statement of Human and Animal Rights: All procedures in this study were conducted in accordance with the Ethics Committee of our institutions.
Statement of Informed Consent: All the patients provided informed consent for inclusion in the study.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.
ORCID iDs: Tomoyuki Matsumoto 
https://orcid.org/0000-0003-4587-0029
Satoshi Sobajima
https://orcid.org/0000-0003-3829-7084
References
- 1. Lawrence RC, Felson DT, Helmick CG, Arnold LM, Choi H, Deyo RA, Gabriel S, Hirsch R, Hochberg MC, Hunder GG, Jordan JM, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II. Arthritis Rheum. 2008;58(1):26–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Buckwalter JA, Martin JA. Osteoarthritis. Adv Drug Deliv Rev. 2006;58(2):150–67. [DOI] [PubMed] [Google Scholar]
- 3. Fodor PB, Paulseth SG. Adipose derived stromal cell (ADSC) injections for pain management of osteoarthritis in the human knee joint. Aesthet Surg J. 2016;36(2):229–36. [DOI] [PubMed] [Google Scholar]
- 4. Song Y, Zhang J, Xu H, Lin Z, Chang H, Liu W, Kong L. Mesenchymal stem cells in knee osteoarthritis treatment: a systematic review and meta-analysis. J Orthop Transl. 2020;24:121–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Bora P, Majumdar AS. Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther. 2017;8(1):145. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, Kloster A, Di Halvorsen Y, Ting JP, Storms RW, Goh B, et al. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells. 2006;24(5):1246–53. [DOI] [PubMed] [Google Scholar]
- 7. Fraser JK, Schreiber RE, Zuk PA, Hedrick MH. Adult stem cell therapy for the heart. Int J Biochem Cell Biol. 2004;36(4):658–66. [DOI] [PubMed] [Google Scholar]
- 8. van Dijk A, Naaijkens BA, Jurgens WJ, Nalliah K, Sairras S, van der Pijl RJ, Vo K, Vonk AB, van Rossum AC, Paulus WJ, van Milligen FJ, et al. Reduction of infarct size by intravenous injection of uncultured adipose derived stromal cells in a rat model is dependent on the time point of application. Stem Cell Res. 2011;7(3):219–29. [DOI] [PubMed] [Google Scholar]
- 9. You D, Jang MJ, Kim BH, Song G, Lee C, Suh N, Jeong IG, Ahn TY, Kim CS. Comparative study of autologous stromal vascular fraction and adipose-derived stem cells for erectile function recovery in a rat model of cavernous nerve injury. Stem Cells Transl Med. 2015;4(4):351–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Domergue S, Bony C, Maumus M, Toupet K, Frouin E, Rigau V, Vozenin MC, Magalon G, Jorgensen C, Noël D. Comparison between stromal vascular fraction and adipose mesenchymal stem cells in remodeling hypertrophic scars. PLoS One. 2016;11(5):e0156161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Shimizu M, Matsumine H, Osaki H, Ueta Y, Tsunoda S, Kamei W, Hashimoto K, Niimi Y, Watanabe Y, Miyata M, Sakurai H. Adipose-derived stem cells and the stromal vascular fraction in polyglycolic acid-collagen nerve conduits promote rat facial nerve regeneration. Wound Repair Regen. 2018;26(6):446–55. [DOI] [PubMed] [Google Scholar]
- 12. Biazzo A, D’Ambrosi R, Masia F, Izzo V, Verde F. Autologous adipose stem cell therapy for knee osteoarthritis: where are we now? Phys Sportsmed. 2020;48(4):392–99. [DOI] [PubMed] [Google Scholar]
- 13. Tsubosaka M, Matsumoto T, Sobajima S, Matsushita T, Iwaguro H, Kuroda R. The influence of adipose-derived stromal vascular fraction cells on the treatment of knee osteoarthritis. BMC Musculoskelet Disord. 2020;21(1):207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Concoff A, Sancheti P, Niazi F, Shaw P, Rosen J. The efficacy of multiple versus single hyaluronic acid injections: a systematic review and meta-analysis. BMC Musculoskelet Disord. 2017;18(1):542. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Giesinger JM, Hamilton DF, Jost B, Behrend H, Giesinger K. WOMAC, EQ-5D and Knee Society score thresholds for treatment success after total knee arthroplasty. J Arthroplasty 2015;30(12):2154–58. [DOI] [PubMed] [Google Scholar]
- 16. Jo CH, Chai JW, Jeong EC, Oh S, Shin JS, Shim H, Yoon KS. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a 2-year follow-up study. Am J Sports Med. 2017;45(12):2774–83. [DOI] [PubMed] [Google Scholar]
- 17. Feng Z, Ting J, Alfonso Z, Strem BM, Fraser JK, Rutenberg J, Kuo HC, Pinkernell K. Fresh and cryopreserved, uncultured adipose tissue-derived stem and regenerative cells ameliorate ischemia-reperfusion-induced acute kidney injury. Nephrol Dial Transplant. 2010;25(12):3874–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Aguiar LT, Martins JC, Brito SAF, Mendes CLG, Teixeira-Salmela LF, Faria CDCM. Knee extensor muscles strength indicates global lower-limb strength in individuals who have suffered a stroke: a cross-sectional study. Braz J Phys Ther. 2019;23(3):221–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods. 2009;41(4):1149–60. [DOI] [PubMed] [Google Scholar]
- 20. Hong Z, Chen J, Zhang S, Zhao C, Bi M, Chen X, Bi Q. Intra-articular injection of autologous adipose-derived stromal vascular fractions for knee osteoarthritis: a double-blind randomized self-controlled trial. Int Orthop. 2019;43(5):1123–34. [DOI] [PubMed] [Google Scholar]
- 21. Fujita M, Matsumoto T, Hayashi S, Hashimoto S, Nakano N, Maeda T, Kuroda Y, Takashima Y, Kikuchi K, Anjiki K, Ikuta K, et al. Paracrine effect of the stromal vascular fraction containing M2 macrophages on human chondrocytes through the Smad2/3 signaling pathway. J Cell Physiol. 2022;237(9):3627–39. [DOI] [PubMed] [Google Scholar]
- 22. Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm. 2014;561459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Zhang L, Li Y, Guan CY, Tian S, Lv XD, Li JH, Ma X, Xia HF. Therapeutic effect of human umbilical cord-derived mesenchymal stem cells on injured rat endometrium during its chronic phase. Stem Cell Res Ther. 2018;9(1):36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Shaw B, Darrow M, Derian A. Short-term outcomes in treatment of knee osteoarthritis With 4 bone marrow concentrate injections. Clin Med Insights Arthritis Musculoskelet Disord. 2018;11:117954411878108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Freitag J, Bates D, Wickham J, Shah K, Huguenin L, Tenen A, Paterson K, Boyd R. Adipose-derived mesenchymal stem cell therapy in the treatment of knee osteoarthritis: a randomized controlled trial. Regen Med. 2019;14(3):213–30. [DOI] [PubMed] [Google Scholar]
- 26. Thirumala S, Gimble JM, Devireddy RV. Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium. J Tissue Eng Regen Med. 2010;4(3):224–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Kamenaga T, Kuroda Y, Nagai K, Tsubosaka M, Takashima Y, Kikuchi K, Fujita M, Ikuta K, Anjiki K, Maeda T, Nakano N, et al. Cryopreserved human adipose-derived stromal vascular fraction maintains fracture healing potential via angiogenesis and osteogenesis in an immunodeficient rat model. Stem Cell Res Ther. 2021;12(1):110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Bastick AN, Belo JN, Runhaar J, Bierma-Zeinstra SM. What are the prognostic factors for radiographic progression of knee osteoarthritis? A meta-analysis. Clin Orthop Relat Res. 2015;473(9):2969–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Belo JN, Berger MY, Reijman M, Koes BW, Bierma-Zeinstra SM. Prognostic factors of progression of osteoarthritis of the knee: a systematic review of observational studies. Arthritis Rheum. 2007;57(1):13–26. [DOI] [PubMed] [Google Scholar]
- 30. Otsuki S, Nakajima M, Lotz M, Kinoshita M. Hyaluronic acid and chondroitin sulfate content of osteoarthritic human knee cartilage: site-specific correlation with weight-bearing force based on femorotibial angle measurement. J Orthop Res. 2008;26(9):1194–98. [DOI] [PubMed] [Google Scholar]
- 31. Kijowski R, Blankenbaker DG, Munoz Del Rio A, Baer GS, Graf BK. Evaluation of the articular cartilage of the knee joint: value of adding a T2 mapping sequence to a routine MR imaging protocol. Radiology. 2013;267(2):503–13. [DOI] [PubMed] [Google Scholar]
- 32. Ronga M, Angeretti G, Ferraro S, DE Falco G, Genovese EA, Cherubino P. Imaging of articular cartilage: current concepts. Joints. 2014;2(3):137–40. [DOI] [PMC free article] [PubMed] [Google Scholar]