Stromal Vascular Fraction and Mesenchymal Stem Cells from Human Adipose Tissue: A Comparison of Immune Modulation and Angiogenic Potential.

INTRODUCTION: In recent years, both stromal vascular fraction (SVF) from adipose tissue and mesenchymal stem cells (MSC) from adipose tissues were extensively used in both preclinical and clinical treatment for various diseases. Some studies reported differences in treatment efficacy between SVFs and MSCs in animals as well as in humans. Therefore, this study is aimed to evaluate the immune modulation and angiogenic potential of SVFs and MSCs from the same SVF samples to support an explanation when SVFs or MSCs should be used. METHODS: The adipose tissue samples from ten female donors with consent forms were collected. SVFs from these samples were isolated according to the published protocols. The existence of mesenchymal cells that positive with CD44, CD73, CD90, and CD105 and endothelial progenitor cells that positive with CD31 and CD34 was determined using flow cytometry. Three samples of SVFs with similar percentages of mesenchymal cell portion and endothelial progenitor cell portion were used to isolate MSCs. Obtained MSCs were confirmed as MSCs using the ISCT minimal criteria. To compare the immune modulation of SVF and MSCs, the mixed lymphocyte assay was used. The lymphocyte proliferation, as well as IFN-gamma and TNF-alpha concentrations, were determined. To compare the angiogenic potential, the angiogenesis in quail embryo assay was used. The angiogenesis efficacy was measured based on the vessel areas formed in the embryos after 7 days. RESULTS: The results showed that all SVF samples contained the portions of mesenchymal cells and endothelial progenitor cells. MSCs from SVFs meet all minimal criteria of MSCs that suggested by ISCT. MSCs from SVFs efficiently suppressed the immune cell proliferation compared to the SVFs, especially at ratios of 1:4 (1 MSCs: 4 immune cells). MSCs also inhibited the IFN-gamma and TNF-alpha production more efficiently than SVFs (p < 0.05). However, in quail embryo models, SVFs triggered the angiogenesis and neovessel formation better than MSCs with more significant vessel areas after 7 days (p < 0.05). CONCLUSION: This study suggested that SVFs and MSCs have different potentials for immune modulation and angiogenesis. SVFs help the angiogenesis better than MSCs, while MSCs displayed the more significant immune modulation. These results can guide the usage of SVFs or MSCs in disease treatment.

Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications.

Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC-MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC-MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1-2 mm(2)) of UC membrane and Wharton's jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic-antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC-MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC-MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC-MSCs cultured in DMEM/F12 plus 1 % antibiotic-antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC-MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC-MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC-MSCs that satisfy the minimum standards for clinical applications.

A Simplified Double-Bundle Anterior Cruciate Ligament Reconstruction by the Three-Inside Technique With Two Suspension Buttons and One Interference Screw.

In comparison with the single-bundle technique, double-bundle anterior cruciate ligament (ACL) reconstruction has proven its superiority regarding biomechanical studies and clinical outcomes in both rotational knee stability and anterior translation function. However, the complexity and risk of complications remain a great concern for the orthopaedic surgeon performing double-bundle ACL reconstruction. We present a simplified double-bundle ACL reconstruction by the 3-inside technique with 2 suspension buttons and 1 interference screw. The semitendinosus tendon is tripled to be the anteromedial (AM) bundle, whereas the gracilis is doubled for the posterolateral (PL) bundle. We perform a 3-socket approach with an inside-out femoral tunnel for the AM bundle, an outside-in femoral tunnel for the PL bundle, and a retrograde tibial socket for the tibial bundle. Thus, this technique is, simply, a combination of 2 procedures: one single all-inside method (for the AM bundle) and one outside-in method (for the PL bundle), with which most arthroscopic surgeons are familiar. The AM and PL bundles are fixed at 30° and 45°, respectively, using 2 suspension buttons and 1 interference screw. Our simplified technique could reduce surgical costs and minimize complications while maintaining isometric position and appropriate graft size for each patient.

Time- and Kellgren⁻Lawrence Grade-Dependent Changes in Intra-Articularly Transplanted Stromal Vascular Fraction in Osteoarthritic Patients.

Knee osteoarthritis (OA) is one of the most prevalent disorders in elderly population. Among various therapeutic alternatives, we employed stromal vascular fraction (SVF), a heterogeneous cell population, to regenerate damaged knee cartilage. OA patients were classified on the basis of age, gender, body mass index (BMI), and x-ray-derived Kellgren-Lawrence (KL) grade. They were treated with SVF and followed-up for 24 months. Visual analogue scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index were used to determine treatment efficacy. Cartilage healing was assessed using the MRI-based Outerbridge score (OS) and evaluation of bone marrow edema (BME) lesions, while a placebo group was used as a control. Time- and KL-dependent changes were also monitored. We observed a decreasing trend in VAS score and WOMAC index in the SVF-treated group up to 24 months, as compared with the placebo group. Besides, a significant increase and decrease in Lysholm and OS, respectively, were observed in the treatment group. Compared with the values before treatment, the greatly reduced WOMAC scores of KL3 than KL2 groups at 24 months, indicate more improvement in the KL3 group. Highly decreased BME in the treated group was also noted. In conclusion, the SVF therapy is effective in the recovery of OA patients of KL3 grade in 24 months.

Comparative Clinical Observation of Arthroscopic Microfracture in the Presence and Absence of a Stromal Vascular Fraction Injection for Osteoarthritis.

Osteoarthritis (OA) is a degenerative cartilage disease that is characterized by a local inflammatory reaction. Consequently, many studies have been performed to identify suitable prevention and treatment interventions. In recent years, both arthroscopic microfracture (AM) and stem cell therapy have been used clinically to treat OA. This study aimed to evaluate the clinical effects of AM in the presence and absence of a stromal vascular fraction (SVF) injection in the management of patients with OA. Thirty patients with grade 2 or 3 (Lawrence scale) OA of the knee participated in this study. Placebo group patients (n = 15) received AM alone; treatment group patients (n = 15) received AM and an adipose tissue-derived SVF injection. The SVF was suspended in platelet-rich plasma (PRP) before injection into the joint. Patient groups were monitored and scored with the Western Ontario and McMaster Universities Arthritis Index (WOMAC), Lysholm, Visual Analog Pain Scale (VAS), and modified Outerbridge classifications before treatment and at 6, 12, and 18 months post-treatment. Bone marrow edema was also assessed at these time points. Patients were evaluated for knee activity (joint motion amplitude) and adverse effects relating to surgery and stem cell injection. Treatment efficacy was significantly different between placebo and treatment groups. All treatment group patients had significantly reduced pain and WOMAC scores, and increased Lysholm and VAS scores compared with the placebo group. These findings suggest that the SVF/PRP injection efficiently improved OA for 18 months after treatment. This study will be continuously monitored for additional 24 months. Stem Cells Translational Medicine 2017;6:187-195.