Transplantation of Nonexpanded Adipose Stromal Vascular Fraction and Platelet-Rich Plasma for Articular Cartilage Injury Treatment in Mice Model

Stromal vascular fraction (SVF) combined with platelet-rich plasma (PRP) is commonly used in preclinical and clinical osteoarthritis as well as articular cartilage injury treatment. However, this therapy has not carefully evaluated the safety and the efficacy. This research aims to assess the safety and the efficacy of SVF combined with PRP transplantation. Ten samples of SVFs and PRPs from donors were used in this research. About safety, we evaluate the expression of some genes related to tumor formation such as Oct-4, Nanog, SSEA3, and SSEA4 by RT-PCR, flow cytometry, and tumor formation when injected in NOD/SCID mice. About efficacy, SVF was injected with PRP into murine joint that caused joint failure. The results showed that SVFs are negative with Oct-4, Nanog, SSEA-3, and SSEA-4, as well as they cannot cause tumors in mice. SVFs combined with PRP can improve the joint regeneration in mice. These results proved that SVFs combined with PRP transplantation is a promising therapy for articular cartilage injury treatment.


Introduction
Stem cell therapy is considered as a promising therapy for degenerative disease treatment, especially articular cartilage injury as well as osteoarthritis. Osteoarthritis was treated by stem cell transplantation for a few years ago. Stem cells from various sources were used to treat this disease. However, the mesenchymal stem cells (MSCs) are considered as most suitable candidates. MSCs are multipotential cells capable of differentiation into bone, cartilage, fat, and some other cells [1]. MSCs could be isolated from bone marrow [2], adipose tissue [3], cord blood [4], banked umbilical cord blood [5], umbilical cord [6], Wharton's jelly [7], placenta [8], and pulp [9]. However, MSCs from bone marrow [10][11][12] and from adipose tissue [13][14][15] are two common stem cell sources for treating cartilage degeneration.
Cartilage degeneration or cartilage injury is a common clinical problem and easily leads to osteoarthritis.
Osteoarthritis is a chronic degenerative process characterized by the degeneration of cartilage, bone bud formation, cartilage reorganization, joint erosion, and loss of joint function [16]. Currently, cartilage injury was treated primarily with drugs [17][18][19][20] or injection of hyaluronic acid [21,22] to reduce the symptoms, pain, and in�ammation control. However, these therapies's efficiencies were limited and oen failed to prevent the degeneration of the joints [23].
Among all of ADSC transplantation cases, SVF is used as noncultured ADSC (nonexpanded ADSC). SVF transplantation has some advantages such as saving time (from isolation to transplant faster about 2-3 hours), being inexpensive, and reducing the risk of cell culture. Although many studies have demonstrated the bene�ts of SVF/ADSC transplantation in cartilage injury treatment, especially knee articular cartilage, so far a little comprehensive studies aim to evaluate the safety and efficiency of SVF transplantation for articular cartilage treatment. erefore, this study aims to evaluate the safety and efficiency of SVF transplantation combined with PRP in the treatment of cartilage injury in the mouse model.

SVF and PRP Preparation.
Firstly, adipose tissue was collected from abdominal fat tissue of ten consenting healthy donors. About 40-80 mL of fat was collected by syringe and stored in 100 mL sterile bottle. Fat was kept at 2-8 ∘ C and then quickly moved to the laboratory. SVF cells are separated from the fat using the extraction kits (Adistem, Australia) according to the manufacturer's guideline. Brie�y, fat is washed 3 times with saline solution to eliminate red blood cells. en, the fat was incubated with a solution AdiExtract (Adistem, Australia). e sample was centrifuged to collect SVF as pellet at the bottom of the tube. To prepare plateletrich plasma (PRP), 50 mL of peripheral blood was taken from a large vein (arm veins). Blood was centrifuged 1,700 rpm for 10 minutes to get platelet-enriching plasma. is plasma is activated with activator solution (Adistem, Australia). en, PRP was mixed with SVF to make the cell suspension. Finally, this suspension was stimulated by the LED light (light monochromatic low energy, Adlight, Adistem, Australia) for 30 minutes before using for treatment.

�uanti�cation of �ucleated Cells from SVF.
Cell suspension (SVF and PRP) is used to count the nucleated cells. Cell number and percentage of viable cells were determined by automatically nucleus-based cell counter (NucleoCounter, Chemometec). Total cell numbers were counted aer permeabilization of the membrane by Reagent A (lysis buffer, Chemometec) and neutralized with a solution of Reagent B (Neutralized buffer, Chemometec). Cell suspension is loaded into the counting chamber containing Propidium iodide dye. For counting dead cells, suspension cells were mixed with only Reagent B solution and loaded into the counting chamber. Survival rate is calculated as follows: (total cell number − the number of dead cells): the total number of cells × 100%.

Evaluation of the Existence of ADSC in SVF.
e existence of ADSC in SVF determined by �ow cytometry. e process summarized as follows: cells were washed twice in physiological saline of Dulbecco-modi�ed PBS (D-PBS) supplemented with 1% bovine serum albumin (Sigma-Aldrich, St Louis, MO). Cells were stained for 30 min at 4 ∘ C with the monoclonal antibody anti-CD44-PE, anti-CD90-PE, and anti-CD105-FITC (BD Biosciences, Franklin Lakes, New Jersey offers). Stained cells were analyzed by �ow cytometer FACSCalibur machine (BD Biosciences). Isotype control is used for all analyzes.

In Vivo Tumorigenicity
Assay. e tumorigenicity of ADSC was evaluated in mice NOD/SCID (NOD.CB17-Prkdcscid/J, Charles River Laboratories). All mice manipulation was according to guideline of laboratory and approved by the Local Ethics Committee of Stem Cell Research and Application, University of Science (VNU-HCM, VN). All mice were kept in clean condition. Mice were injected subcutaneously at a concentration of 10 5 , 10 6 , and 10 7 cells, respectively in three groups (each group with 3 mice). Control group was injected with PBS. e formation of tumors in mice was followed for 3 months.

Articular Cartilage Injured Mice Model and Experimental Treatment Schedule.
To evaluate the efficiency of SVF transplantation in articular cartilage injury, we used articular cartilage injured mouse model. e NOD mouse/SCID mice were anesthetized with ketamine (40 mg/kg), then joint destruction by �ne needle 32.5 G. Normal mice were used as a positive control (uninjured). Nine mice randomly divided into the treatment group (5 mice) and negative control group (4 mice). Six hours aer injury, the mice were treated. In the treatment group, 200 L containing 2 ⋅ 10 6 SVF in PRP (the treatment group) or PBS (the negative control group) was injected into the knee joint via two doses, with a 10 min interval between injections.
Mice were recorded some parameters related to joint regeneration for 45 days. e mice were recorded the movement on the table daily. At the 45th day, all mice were anesthetized, and their hind limbs were cut and used for

Results and Discussion
MSCs have the large differentiative potential, easily differentiate into bone, cartilage, and adipocyte. Autologous MSC transplantation is considered as a safe and effective therapy in some patients. Recently, adipose tissue was identi�ed as the abundant source of MSCs. ADSCs exist with large amounts of adipose tissue [38]. Similar to MSCs from other sources, ADSC have the ability to differentiate into fat cells, bone, and cartilage and transdifferentiate into neurons and muscle [39][40][41][42][43][44][45]. erefore, ADSC is favored as a source of autologous cell transplantation. However, the isolated ADSC relatively complex, consuming time, so ADSCs were mainly used as SVF (containing ADSC) without culture. is study aims to evaluate the safety and efficiency of SVF transplantation resuspended in PRP in mouse model. In the �rst experiment, we successfully isolated SVF and PRP. Compared to other studies, we have successfully isolated 0.32 ± 0.15 × 10 6 SVF cells from 1 gram of fat with a survival rate of 90.90% ± 8.57% ( 10). Next, we assessed the existence of ADSC or MSC in the SVF. Analysis results from 10 samples showed that ADSCs existed in all samples. ADSC counted to 0.89% ± 0.11% in the SVF. ADSC populations were identi�ed based on the expression of CD44, CD90, and CD105 of them (Figure 1). ese results were similar to many other authors on ADSC markers [43,[46][47][48][49][50][51][52]. e markers satis�ed the criteria of MSC following to Dominici et al. (2006) [53].
To assess safety, we have evaluated the expression of genes related to cancer. In particular, two genes Oct-3/4 and Nanog were assessed by real-time RT-PCR method and SSEA-3, and SSEA-1 was assessed by �ow cytometry. e results showed expression of Oct-3/4, Nanog, SSEA-3, and SSEA-1 much lower than embryonic stem cells (Figure 2). ese results demonstrated that the SVF hold low tumorigenicity. In fact, (e) (f) F 3: HE staining of articular cartilage. Mature cartilage layer was recorded in normal mouse (a). Mature cartilage was thinned by needle (b). Injured cartilage was regenerated in negative control group (c, e) and treated group (d, f). However, the neocartilage in treated group was thicker than in negative control group.
Nanog and Oct-3/4 participate in the process of self-renewal of embryonic stem cells [54,55]. Moreover, these proteins related to the tumorigenicity process in mature germ cells [56], carcinoma oral squamous cell [57], lung cancer [58], breast cancer [59], and gliomas [60]. e SVF and PRP injection under the skin mouse NOD/SCID could not form teratomas. With these experiments, we concluded that the SVF plus PRP has a promising therapy with a high safety for transplantation experiments.
In the next experiment, we evaluated the efficiency of SVF transplantation in articular cartilage injury. e results showed that the SVF plus PRP transplantation signi�cantly improved the articular cartilage injury compared to control. In the treated group, mice exhibited a reduction of the time required that mice could move on the table by injured hind limb compared to control. In the control group, mice can move by injured legs aer 38.5 ± 4.30 days, while in the treated group, mice could move by injured legs aer 29.4 ± 4.32 days.
About histological analysis, in the treated group, an average area of the cartilage damage was 62.60%, and there was 35.5% of neocartilage formation aer 45 days ( 5). While in the control group, average area of cartilage lesions was 53.13%, but only 15.5% of neocartilage formation aer 45 days (Figure 3). e grade of cartilage injury between two experimental groups was different due to the effects of the dissimilar force from needle. Aer 45 days, results showed that 35.5% of neocartilage formed in treated group, while only 15.5% of neocartilage formed in the negative control group. is suggested that SVF and PRP gave bene�t effects on the enhancement as well as trigger the neocartilage forming. More importantly, the articular cartilage in both of groups completed at the same level aer 45 days with 12 cell layers. ese results demonstrated that the SVF and PRP could participate in the process of self-renewal of joint cartilage at the joint microenvironment. Especially, there were no scar tissues or tumors forming at the gra sites. is result was similar to the previous publications about SVF plus PRP transplantation in the treatment of cartilage injury in dog [31][32][33], rabbits [34,61], horses [14,61], rat [35], mice [36], and goats [37]. For example, in joint injured mice model by collagenase, Ter Huurne et al. (2011) showed that the level of damage nearly 50% reduction in ADSC transplanted mice compared to control aer 42 days [36]. Speci�cally, knee injury went down to 25% in treated mice compare to 88% in controls. ey suggested that the transplanted ADSC protected and healed of injured cartilage [37]. e �ndings of Dragoo et al. (2007) showed that autologous ADSCs could reestablish the joint surface in rabbits, in which 100% of rabbits (12/12) had the occurrence of neocartilage, while only 8% rabbit (1/12) in the control had the appearance of neocartilage ( ) [62]. Roles of SVF or ADSCs in cartilage regeneration were recorded with many different effects. In fact, similar to MSCs derived from bone marrow, ADSC had anti-in�ammatory properties [63,64] and inhibition of gra versus host disease (GVHD) [65]. e transplantation of ADSC could successfully treat gra versus host disease with steroid-resistant form [66,67]. All of these roles could add more effects to trigger rapid cartilage regeneration in this study.
Besides, in this study, ingredients from PRP also had important roles in stimulated graed cells as well as endogenous cells growth and differentiation. ere are at least six known growth factors such as platelet-derived growth factor (PDGF) that promotes blood vessel growth, cell division, and forming the skin; transforming growth factor-beta (TGF-) that promotes cell division mitosis and bone metabolism; vascular endothelial growth factor (VEGF) that promotes the blood vessel formation; epidermal growth factor (EGF) that promotes cell growth and differentiation, angiogenesis, and collagen formation; �broblast growth factor-2 (FGF-2) that promotes the growth of cell differentiation and angiogenesis; and insulin-like growth factor (IGF) that is a regulator in all cell types of the body [68,69]. PRP injection also showed that improvements in knee injury and osteoarthritis score, including pain and symptom relief [70,71].
Combining effect of SVF and PRP has a positive effect on the stimulation of proliferation, differentiation, and regeneration of cartilage in a mouse model. However, SVF also has a few limitations, notably the relatively low presence of ADSCs in the SVF. erefore, SVF cultured to enrich ADSC before being transplanted may be essential, especially a little obtained fat cases.

Conclusion
Adipose tissue provides a rich source of MSCs. e SVF and PRP injection are a promising therapy in injured articular cartilage regeneration. is therapy signi�cantly improved the injured articular cartilage. However, this study only assesses the ability of tumorigenicity and efficiency in mouse. Some side effects such as fever and muscle pain as well as the tumorigenicity in human being when using SVF and PRP could not be checked in this research.