Clinical and Magnetic Resonance Image Changes of Platelet-Rich Plasma Therapy in Combination with Human Mesenchymal Stem Cells from Autologous Adipose Tissue for Knee Osteoarthritis Treatment

Objective: To evaluate the efficacy based on clinical symptom and on magnetic resonance image of platelet-rich plasma therapy in combination with mesenchymal stem cells from autologous adipose tissue for knee osteoarthritis treatment. Patients and Method: 30 patients including 26 females and 4 males; correspondingly, 60 knee joints were diagnosed with osteoarthritis with stages II - III of Kellgren and Lawrence, their mean age was 58.63 ± 11.11. All were injected with autologous platelet-rich plasma that was extracted by PRP set, APC 30 PRP PRCEDURE PRAK and autologously extracted mesenchymal stem cells from abdominal adipose tissue using the ADI-25-01 ADIPOSEPRCEDURE PRAK of USA. Results: After 12 months: the pain level according to VAS score at the right knee joint was decreased from 6.0 ± 1.28 before treatment to 1.9 ± 0.3; VAS score at the left knee joint was decreased from 6.43 ± 1.19 to 2.25 ± 0.43. Total Lequene score at right knee joint was decreased from 16.04 ± 1.57 before treatment to 4.31 ± 1.04, at left knee joint was decreased from 17.52 ± 1.74 before treatment to 5.15 ± 1.48. Total WOMAC score at right knee joint was decreased from 55.93 ± 5.56 to 10.37 ± 1.56; at left knee joint was decreased from 53.97 ±


Introduction
Osteoarthritis of the knee is a degenerative disease that can be classified into primary and secondary knee osteoarthritis. Primary knee osteoarthritis is a disease of unknown cause which is mainly related to the aging process. Secondary knee osteoarthritis with identifiable etiology includes endocrine disorders, anatomical abnormalities, post-traumatic arthritis, and septic arthritis [1]. Currently, the treatment of knee osteoarthritis is limited. There are several conventional therapies for knee osteoarthritis which include physical therapies, non-steroidal anti-inflammatory pain relievers, drugs which slow down the degenerative process such as glucosamine, joint lubricant supplements such as hyaluronic acid, platelet-rich plasma (PRP) or intra-articular corticosteroid injections, traditional medicine measures and arthroscopic knee surgery [2] [3]. All of the above-mentioned treatments can only relieve symptoms which do not restore articular cartilage. When degeneration is more severe, total knee replacement surgery is required [2] [3].
Autologous intra-articular platelet-rich plasma (PRP) injection is a new method in the treatment of osteoarthritis. Platelet-rich plasma has been shown to contain growth factors, in which, TGF-β (Transforming Growth Factor-β) plays an important role to increase the base for chondrocytes' growth, the proliferation of chondrocytes and the regulation of proteoglycan synthesis [4].
In the world, in the past few years, many studies have shown that autologous intra-articular PRP injection is an effective therapy in the treatment of knee osteoarthritis [5]. This has been proven by many studies around the world.
Mesenchymal stem cells (MSCs) have the potential for self-renewal and multidirectional differentiation [6] which can exert therapeutic effects on various diseases through directed differentiation [7], regulate immune system [8], anti-inflammatory, progenitor [9], improve microenvironment [10] and promote regeneration [10]. MSCs have been used in the treatment of various diseases [11], such as ovarian failure, Parkinson's disease, nervous system damage and amyotrophic lateral sclerosis (ALS). MSCs therapy can be applied in the treatment of knee osteoarthritis and has shown encouraging results [7] [11].
Recent researches have shown the presence of stem cells in adipose tissues which are known as adipose-derived stem cells (ADSCs). These cells are referred to as mesenchymal stem cells (MSCs) that expose a number of special characteristics. They participate in the fibroblast-like surface formation and differentiate Open Journal of Regenerative Medicine into osteoblasts, cartilage, and adipocytes [12]. Many researchers have been conducted over the last few years which include preclinical and clinical trials to perform the treatment of cartilage injuries and knee osteoarthritis.
Stem cell therapy is a milestone in regenerative medicine for the treatment of knee osteoarthritis. MSCs have not been widely adopted because of cell source problems and expensive cell cultures. Furthermore, its efficacy and safety are being explored [13].
Therefore, the combination therapy of platelet-rich plasma with mesenchymal stem cells from autologous adipose tissue will promote both types of effects: 1) TGF-β plays the roleincreasing the substrate for chondrocyte's growth, proliferation of chondrocytes, regulation of proteoglycan synthesis; 2) mesenchymal stem cells increase cartilage regeneration, repair the organization of damaged cartilage, regenerate subchondral bone to heal joints. Therefore, the aim of this study was to evaluate the efficacy of platelet-rich plasma therapy combined with autologous adipose tissue mesenchymal stem cells in the treatment of knee osteoarthritis.

Study Setting and Design
The research is a randomized clinical trial design which evaluates the results by comparing those before and after treatment 1 week, 1 month, 3 months, 6 months and 12 months according to clinical rating scales and changes of cartilage thickness on magnetic resonance images.
The study was implemented at Vinh Medical University Hospital, from June 2020 to February 2021.

Study Population
In the study, there are selected 30 patients including 26 females and 4 males; correspondingly, 60 knee joints were diagnosed with osteoarthritis according to the American College of Rheumatology (ACR) [14] who are at the mean age of 58.63 ± 11.11, disease duration is from 5.3 ± 4.6 years which.
The inclusion criteria: -Patients were diagnosed the osteoarthritis at stages II -III according to the classification standard of Kellgren and Lawrence.
The exclusion criteria: the patients were at stage I, stage IV of knee osteoarthritis; have severe anemia and platelets < 150,000/mm 3 , have secondary knee osteoarthritis, post-traumatic, infectious degeneration, cancer or have contraindications to surgery.
Patients received ultrasound and magnetic resonance imaging to evaluate input data before injecting platelet-rich plasma in combination with mesenchymal stem cells from autologous adipose tissue.

Study Materials
Autologous platelet-rich plasma was extracted from 30 ml of peripheral blood according to the process of PRP set, APC 30 PRP PRCEDURE PRAK and the Harvest smart extraction system of TERUMO BCT-USA, following an 8-step process with a count of 5 ml (platelet count is up to 1.5 million/ml). This 5ml platelet-rich plasma was contained in a clean, sterile cup that had ready the activating agent.
Autologous mesenchymal stem cells were obtained from the patient's abdominal adipose tissue and was extracted by a set of ADI-25-01 ADIPOSEPRCEDURE PRAK (2 syringes, 25 ml in each syringe) and by the Harvest Smart Stem Cell Extractor of TERUMO BCT-USA company according to the 13-step process, the obtained count is 10 ml with 1.6 million cells.
Two products with the platelet-rich plasma and the mesenchymal stem cells were mixed together in a clean, sterile cup (15 ml including: 5 ml of the platelet-rich plasma and 10 ml of the mesenchymal stem cells) and then were equally divided into 2 single-use syringes, each 7.5ml syringe was injected into a knee joint.
All procedures are carried out in the operating room, extraction room, and sterile injection room.

Statistical Analysis
The data was processed using SPSS 20.0 software; qualitative variables are expressed as frequency and proportions, quantitative variables are expressed as mean ± standard deviation. The Chi 2 test was used to compare the 2 proportions and the T test was to compare the 2 means. The difference was statistically significant when p < 0.05.

Baseline Characteristics
In our study sample, the number of male patients including 4 people accounted for 13.3%. The number of female patients accounted for 86.7% and the number of knee osteoarthritis with stage II was 20% and stage III damage accounted for 80%. Baseline characteristics of patients were summarized in Table 1.

Evaluation of Treatment Results through the VAS Scale
The average VAS pain score of right knee before treatment was 6 ± 1.28 which decreased to 2.70 ± 0.46 after 6 months of treatment and to 1.90 ± 0.30 after 12 months of treatment. This improvement was statistically significant with p < 0.001.The mean VAS pain score of the left knee joint before treatment was 6.43 ± 1.19, which decreased to 3.5 ± 0.51 after 6 months of treatment and to 2.25 ± 0.43 after 12 months of treatment. This improvement was statistically significant with p < 0.001.The results were presented in Table 2. The changes Lequesne scale after treatment 12 months versus before treatment were showed in Table 3.

Evaluation of Treatment Results by WOMAC Scale
Overall WOMAC scale of right knee joint was decreased from 55.93 ± 5.56 to 10.37 ± 1.56 after 12 months of treatment. There was a remarkable improvement in the scales of pain WOMAC, mobility WOMAC, and stiffness WOMAC after the treatment. Results were presented in Table 4.
Overall WOMAC scale of left knee joint was decreased from 53.97 ± 5.57 to 10.07 ± 1.56 after 12 months of treatment. There was a remarkable improvement in the scales of pain WOMAC, mobility WOMAC, and stiffness WOMAC after the treatment. Results were showed in Table 5.

Evaluation of the Results Based on the Change of Cartilage Thickness on Magnetic Resonance Image
The surface of articular cartilage on magnetic resonance in the above mentioned positions were improved after 12 months of treatment, the difference was statistically significant. The changes of cartilage thickness on magnetic resonance were showed in Table 6.
After 12 months of treatment, there were 8 joints (13.33%) that did not change the total thickness of cartilage at the measured locations (lower end of femur, upper end of tibial, patella joint), found in degenerative joints of stage III.
Results were presented in Table 7.

Clinical Evaluation of Treatment Results
After 12 months of treatment with a mixture of platelet-rich plasma in combination with mesenchymal stem cells from autologous adipose tissue, we found that the patients had reduced pain, improved motor function, and improved signs of knee stiffness which was as follows.   The patient's joint pain relief was shown by the mean of VAS score at the right knee, the left knee joint decreased significantly compared to before treatment. This improvement started after 1 month, 6 months and was most pronounced after 12 months.
Patients improved signs of stiffness, the ability to walk, go up and down stairs, squat as shown by the mean of LEQUESNE score of the right knee and the left knee all decreased significantly compared to before treatment with p < 0.001.
The WOMAC score of both right and left knee decreased significantly after 12 months of treatment. In which, there was a significant improvement in the WOMAC pain scale, WOMAC movement score, and WOMAC joint stiffness score after the treatment.
In this regard, some studies also showed similar results.
In the study by Bui et al.

Evaluation of Treatment Outcomes Based on MRI
The results of our study depicted that all positions of the surface of articular cartilage on MRI has improved. Specially, the lateral condyle of the lower femoral increased from 1.40 ± 0.10 mm before treatment to 1.46 ± 0.11 mm after 12 months of treatment.  (Table 6). After 12 months of treatment, remaining 8 stage-III knees (13.33%) had no change in total cartilage thickness at measurement sites at the lower head of femoral, the upper tibia and the patellar joint (Table 3). Several studies have used parameters on MRI such as the change of cartilage thickness to evaluate the effectiveness of autologous adipose tissue stem cell therapy or platelet-rich plasma therapy in the treatment of knee osteoarthritis.
Study by Tran et al. (2016) showed that there was an improvement in articular cartilage thickness on MRIat 4 positions which were medial tibial plateau, lateral tibial plateau, medial condyle, and lateral condyle of the femur at the time of 6 months and 1 year follow-up but there was no statistical significance (p > 0.05). This may be because the majority of study subjects are mild knee osteoarthritis (stage I-II), so the difference was not significant [17].
Another study by Pham (2017) on treatment of primary osteoarthritis with autologous adipose stem cell therapy, the findings depicted that: the thickness of articular cartilage at the femoral condyle increased from 1.52 ± 0.57 mm before treatment to 1.61 ± 0.59 mm after 6 months and to 1.65 ± 0.56 mm after 1 year. The thickess of articular cartilage at the tibial plateau from 1.59 ± 0.59 mm before treatment to 1.68 ± 0.59 mm after 6 months and to 1.75 ± 0.57 mm after 1 year [16].
In 2012, Yong-Gon Koh and colleagues evaluated the clinical and imaging results in 18 patients with knee osteoarthritis treated with autologous adipose tissue stem cells at the lower-kneecap. The authors evaluated the improvement of articular cartilage by the WORMS (whole organ magnetic resonance imaging score) scale, the findings showed that the WORMS score decreased from 60.0 to 48.3 points (p < 0.01). In particular, this study also showed that there was a posi-Open Journal of Regenerative Medicine tive correlation between the improvement of clinical symptoms and images of knee osteoarthritis with the amount of injected stem cells. The authors concluded that adipose tissue stem cells were a valuable source of cells in the treatment of articular cartilage damage [18] [19].
In 2016, a study by Liang-jing Lu and colleagues on 18 patients with bilateral knee osteoarthritis who were treated with adipose tissue stem cell therapy with 3 injections: before treatment, after 3 weeks and after 48 weeks, the results showed that the volume of articular cartilage of the femur, tibia and patella increased steadily during the entire follow-up period, which was statistically significant after 6 months, 12 months and 18 months [20].
In summary, all above researches have depicted that the adipose tissue stem cells was effective in repairing cartilage damage.

Conclusion
The treatment of knee osteoarthritis by platelet-rich plasma therapy in combination with mesenchymal stem cells from autologous adipose tissue was effective in reducing pain, improving patient's mobility and walking function through scale reductions of VAS, LEQUENE, WOMAC comparing with before treatment, improving significantly the thickness of articular cartilage on magnetic resonance image.