Adipose-derived Stromal Vascular Fraction Research Articles

Stromal Vascular Fraction Reverses the Age-Related Impairment in Revascularization following Injury

Adipose-derived stromal vascular fraction (SVF) has emerged as a potential regenerative therapy, but few studies utilize SVF in a setting of advanced age. Additionally, the specific cell population in SVF providing therapeutic benefit is unknown. We hypothesized that aging would alter the composition of cell populations present in SVF and its ability to promote angiogenesis following injury, a mechanism that is T cell-mediated. SVF isolated from young and old Fischer 344 rats was examined with flow cytometry for cell composition. Mesenteric windows from old rats were isolated following exteriorization-induced (EI) hypoxic injury and intravenous injection of one of four cell therapies: (1) SVF from young or (2) old donors, (3) SVF from old donors depleted of or (4) enriched for T cells. Advancing age increased the SVF T-cell population but reduced revascularization following injury. Both young and aged SVF incorporated throughout the host mesenteric microvessels, but only young SVF significantly increased vascular area following EI. This study highlights the effect of donor age on SVF angiogenic efficacy and demonstrates how the ex vivo mesenteric-window model can be used in conjunction with SVF therapy to investigate its contribution to angiogenesis.

Autologous adipose-derived stromal vascular fraction and platelet concentrates for the treatment of complex perianal fistulas

BackgroundComplex perianal fistulas are a major challenge for modern surgery since 10–35% of patients have functional problems after treatment. Sphincter-saving techniques have a wide range of efficacy (10–80%). We hypothesised that autologous adipose-derived stromal vascular fraction in combination with platelet rich plasma is a new therapeutic strategy with enhanced cure and function preservation rates.MethodsAdult patients with complex cryptoglandular perianal fistulas were treated with injection of autologous adipose-derived stromal vascular fraction in combination with platelet rich plasma around and inside the fistulous tract between May 2018 and April 2019 at the General and Emergency Surgery Operative Unit of the University Hospital “P. Giaccone” of Palermo. Fistulas were confirmed by magnetic resonance imaging. Patients completed the Short Form-36 score on quality of life and the Wexner and Vaizey scores on faecal incontinence, and they were functionally studied using a three-dimensional anorectal manometry. The clinical and functional follow-up was performed at 1 year and 2 years after surgery.ResultsNine patients (4 males, 5 females; median age 42 years [19–63 years]) with high trans-sphincteric or horseshoe fistulas were treated. The average number of previous surgeries per patient was 4.8. At 1 year follow-up, 77.7% of patients were cured, while at 2 years there was 1case of relapse. The variation in Short Form-36 score in cured patients was not significant (p = 0.0936). No statistically significant differences were found in continence scores.ConclusionsThe proposed treatment is a treatment option that preserves sphincter integrity and function, potentially avoiding postoperative incontinence and the need of repeated treatments.

Adipose stromal vascular fraction reverses mitochondrial dysfunction and hyperfission in aging-induced coronary microvascular disease.

Aging is associated with blunted coronary microvascular vasodilatory function. Previously, systemically administered adipose stromal vascular fraction (SVF) therapy reversed aging-induced attenuation of β1-adrenergic- and flow-mediated dilation dependent on reducing mitochondrial reactive oxygen species. We hypothesized that SVF-mediated recovery of microvascular dilatory function is dependent on recovery of mitochondrial function, specifically by reducing mitochondrial hyperfission. Female Fischer-344 rats were allocated into young control, old control, and old + SVF therapy groups. Pressure myography, immunofluorescent staining, Western blot analysis, and RNA sequencing were performed to determine coronary microvascular mitochondrial dynamics and function. Gene and protein expression of fission-mediator DRP-1 was enhanced with aging but reversed by SVF therapy. SVF facilitated an increase in fusion-mediator MFN-1 gene and protein expression. Mitochondrial morphology was characterized as rod-like and densely networked in young controls, isolated circular and punctate with aging, and less circularity with partially restored mitochondrial branch density with SVF therapy. Decreased mitochondrial membrane potential and ATP bioavailability in aged animals at baseline and during flow-mediated dilation were reversed by SVF and accompanied with enhanced oxygen consumption. Dilation to norepinephrine and flow in young controls were dependent on uninhibited mitochondrial fusion, whereas inhibiting fission did not restore aged microvessel response to norepinephrine or flow. SVF-mediated recovery of β-adrenergic function was dependent on uninhibited mitochondrial fusion, whereas recovery of flow-mediated dilation was dependent on maintained mitochondrial fission. Impaired dilation in aging is mitigated by SVF therapy, which recovers mitochondrial function and fission/fusion balance.NEW & NOTEWORTHY We elucidated the consequences of aging on coronary microvascular mitochondrial health as well as SVF's ability to reverse these effects. Aging shifts gene/protein expression and mitochondrial morphology indicating hyperfission, alongside attenuated mitochondrial membrane potential and ATP bioavailability, all reversed using SVF therapy. Mitochondrial membrane potential and ATP levels correlated with vasodilatory efficiency. Mitochondrial dysfunction is a contributing pathological factor in aging that can be targeted by therapeutic SVF to preserve microvascular dilative function.

Local transplantation of syngeneic adipose stromal vascular fraction ameliorates damaged anal sphincter function in a rat model of vaginal distension

BackgroundThe adipose stromal vascular fraction contains abundant mesenchymal stem cells and is utilized for cell therapy of male stress urinary incontinence. The purpose of this paper was to explore the effect of local transplantation of the stromal vascular fraction on improvement of damaged anal sphincter function. MethodsA rat model of vaginal distension was used as a model of damaged anal sphincter function. The adipose stromal vascular fraction was separated from the inguinal fat of syngeneic green fluorescent protein transgenic rats and delivered into the internal anal sphincter of vaginal distension rats. The maximum resting pressure was evaluated during insertion and withdrawal of the catheter at 4 or 10 days after vaginal distension treatment to estimate anal sphincter function. Green fluorescent protein-transfected human-adipose–derived mesenchymal stem cells were transplanted into the internal anal sphincter of nude rats. Hematoxylin-eosin and Masson trichrome staining were performed to evaluate tissue damage and collagen synthesis. Transplanted cells were identified using a green fluorescent protein antibody and a human-specific antibody. Activation of the transplanted human-ADSC was evaluated by quantitative RT-PCR ResultsThe mean maximum resting pressure (during catheter withdrawal) of vaginal distension rats was significantly lower than that of control rats, and stromal vascular fraction injection normalized it 4 days after treatment (control: 5.66 ± 0.98, vaginal distension: 4.04 ± 1.28, vaginal distension + stromal vascular fraction: 5.92 ± 1.28 [mmHg, control versus vaginal distension: P = .039; vaginal distension versus vaginal distension + stromal vascular fraction: P = .007]). Histological examination showed that vaginal distension disrupted the internal anal sphincter, and the transplanted syngeneic stromal vascular fraction survived for 10 days. Transplanted xenogeneic human-adipose–derived mesenchymal stem cells survived in the internal anal sphincter of nude rats for 4 and 10 days. Genes related to extracellular remodeling were up-regulated in the transplanted human-adipose–derived mesenchymal stem cells ConclusionSyngeneic and heterotopic transplanted adipose-derived mesenchymal stem cells engrafted in the internal anal sphincter and ameliorated damaged anal sphincter function in a rat model of vaginal distension.

Stromal Vascular Fraction Restores Vasodilatory Function by Reducing Oxidative Stress in Aging-Induced Coronary Microvascular Disease.

Aims: The objective of this study is to identify mechanisms for adipose stromal vascular fraction's (SVF) restorative effects on vasodilation in aging-induced coronary microvascular disease (CMD). We hypothesize that reactive oxygen species (ROS) diminish β1-adrenergic receptor (β1ADR)- and flow-mediated dilation (FMD) in coronary arterioles, reversible by SVF and adipose-derived stem cells (ADSCs). Results: SVF attenuates aging-induced chronic accumulation of ROS and pro-oxidant gene and protein expression with enhancement of antioxidant gene and protein expression and glutathione, but not nitric oxide. ADSCs attenuate hydrogen peroxide while restoring nitric oxide and glutathione. Mass spectrometry of SVF- and ADSC-conditioned media reveals abundant antioxidant proteins suggesting a paracrine mechanism. FMD and β1ADR-mediated dilation diminished with aging, restored with SVF and ADSCs. FMD was restored by a switch in the acute signaling mediator from hydrogen peroxide in aging to peroxynitrite with SVF and ADSCs. Vasorelaxation to β1ADR-agonism was mechanistically linked with hydrogen peroxide, nitric oxide, and glutathione. Exogenous ROS eliminates isoproterenol-mediated dilation in youth that is blocked by inhibition of pro-desensitization and internalization proteins while nitric oxide enhances isoproterenol-mediated dilation in aging. Innovation: We introduce a novel mechanism by which ROS impacts β1ADR trafficking: the ROS/RNS-β1ADR desensitization and internalization axis. Aging-induced ROS shunts β1ADR from the plasma membrane into endosomes. SVF reduces oxidative burden, restoring functional β1ADR. Conclusions: SVF (and ADSCs to a lesser extent) reduce oxidative stress, and restore flow- and β1ADR-mediated vasodilation in aging. SVF represents a promising therapeutic strategy for CMD by addressing root cause of pathology; that is, oxidative stress-mediated hyperconstriction. Antioxid. Redox Signal. 38, 261-281.

Adipose stromal vascular fraction: a promising treatment for severe burn injury.

Thermal skin burn injury affects both adults and children globally. Severe burn injury affects a patient's life psychologically, cosmetically, and socially. The pathophysiology of burn injury is well known. Due to the complexity of burn pathophysiology, the development of specific treatment aiding in tissue regeneration is required. Treatment of burn injury depends on burn severity, size of the burn and availability of donor site. Burn healing requires biochemical and cellular events to ensure better cell response to biochemical signals of the healing process. This led to the consideration of using cell therapy for severe burn injury. Adult mesenchymal stem cells have become a therapeutic option because of their ability for self-renewal and differentiation. Adipose stromal vascular fraction (SVF), isolated from adipose tissues, is a heterogeneous cell population that contains adipose-derived stromal/stem cells (ADSC), stromal, endothelial, hematopoietic and pericytic lineages. SVF isolation has advantages over other types of cells; such as heterogeneity of cells, lower invasive extraction procedure, high yield of cells, and fast and easy isolation. Therefore, SVF has many characteristics that enable them to be a therapeutic option for burn treatment. Studies have been conducted mostly in animal models to investigate their therapeutic potential for burn injury. They can be used alone or in combination with other treatment options. Treatment with both ADSCs and/or SVF enhances burn healing through increasing re-epithelization, angiogenesis and decreasing inflammation and scar formation. Research needs to be conducted for a better understanding of the SVF mechanism in burn healing and to optimize current techniques for enhanced treatment outcomes.

Breast Fat Grafting: Comparing Filtration-Centrifugation to Filtration-Washing Fat Processing

Breast Fat Grafting: Comparing 2 fat processing methods and learned clinical experience. Introduction: Since alloplastic breast implants suffer from numerous potential complications and revision surgery, some practitioners and patients seek alternative treatment. Autologous fat grafting has gained popularity due to its safety and scientific advances yielding increased survival. Materials and Methods: Laboratory evaluation of fat specimens compared to ultrasound-assisted and suction-assisted fat harvesting is presented. The filtration-washing device (PureGraft) versus the filtration-centrifugation device (LipoKit) were compared using both clinical evaluation, laboratory analysis, and diagnostic ultrasound to assess fat thickness before and after surgical placement. Results: The study evaluated 12 women electing breast fat transfer, 6 undergoing PureGraft for processing (mean 29.2 years, Mean BMI 25.5), with 3 of these using suction-assisted harvested fat and 3 using ultrasound-assisted harvested fat. The initial group had 6 patients (mean 28.5 years, Mean BMI 25.1) that had undergone LipoKit fat processing, with 3 of these using suction-assisted harvested fat and 3 using ultrasound-assisted harvested fat. Fat placed ranged between 100 cc and 240 cc per breast in the subcutaneous plane, with a mean of 171.7 cc for LipoKit and 169.2 cc for the PureGraft group. LipoKit fat processing revealed fat grafted breasts to be 40% thicker via diagnostic ultrasound at 10 weeks and 29% thicker at a mean of 7 months and 10 days. The ultrasound-assisted fat harvesting using LipoKit found a mean fat viability of 91.5% compared to 81.8% with PureGraft. Suction-assisted fat harvesting using LipoKit found a mean fat viability of 92.1% compared to 91.5% with PureGraft. Discussion: Our clinical study revealed that in the patient population that elected breast fat grafting as the method of augmentation, there was a high patient satisfaction rate. The filtration-centrifugation system has several advantages: (1) centrifugation forces injuries the larger, more mature, less likely to survive adipocytes (2) the process removes the oil content much more efficiently and in greater quantities than PureGraft, (3) more efficiently and in greater quantities extracts the wetting fluid (4) absence of washing inhibits the removal of lipoaspirate containing growth factors and cytokines, (5) Compacts the fat, and finally (6) Concentrates adipose-derived stem cells and stromal vascular fraction progenitor support cells. There was no difference in fat viability comparing suction-assisted to ultrasound-assisted liposuction fat harvesting. Conclusions: Breast fat grafting using the Filtration-Centrifugation device (LipoKit) revealed superior volume results compared to the Filtration-Washing device (PureGraft), with no difference with suction or ultrasound fat harvesting.

Clinical Safety and Effectiveness of Adipose-Derived Stromal Cell vs Stromal Vascular Fraction Injection for Treatment of Knee Osteoarthritis: 2-Year Results of Parallel Single-Arm Trials

Background: There are currently no disease-modifying treatments available for knee osteoarthritis (OA), although cultured adipose-derived stromal cells (ASCs) have shown promise in experimental models. However, given the regulatory limits on the use of cultured cells in humans, previous trials have focused primarily on the stromal vascular fraction (SVF) intra-articular injection. Therefore, the therapeutic value of ASCs for knee OA remains unknown. Purpose: To study ASC versus SVF intra-articular injection in patients with Kellgren-Lawrence (KL) knee OA grades 2 to 4 in parallel single-arm trials. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 80 patients were enrolled, with 42 (72 knees) receiving ASC intra-articular injection and 38 (69 knees) receiving SVF. Patient-reported outcome measures were assessed at 1, 3, 6, 12, and 24 months using the Knee injury and Osteoarthritis Outcome Score 5 (KOOS5) and pain visual analog scale (VAS). The percentages of patients achieving the minimal clinically important difference (MCID) and Patient Acceptable Symptom State (PASS) were also calculated. Per protocol, a subset of the ASC group received an ASC booster injection after 6 months. A repeated-measures analysis of variance compared results between treatment arms and by KL grade over time. Results: Patient-reported outcome measures improved substantially after both treatments (P < .05 at all time points), with the ASC group more likely to achieve the MCID (50% vs 24%; P = .01) and PASS (45% vs 24%; P = .04) for the pain VAS and the MCID (43% vs 16%; P = .02) for the KOOS5 at 12 months, although not at 24 months. Knees treated with ASC for KL grade 2/3 OA had significantly superior outcomes compared with those with KL grade 4 OA for the KOOS5 (P = .01) and pain VAS (P = .03), but no such difference was observed in knees treated with SVF. Three patients receiving ASCs (7%; all KL grade 3) sought additional nonoperative treatment by 24 months versus 9 patients receiving SVF (24%; all KL grade 3) (P = .06). ASC booster injections conferred no additional benefit. Notably, patients in the ASC cohort reported more injection-site pain and swelling after the booster injection than after the initial injection (P < .01). Conclusion: This represents the first head-to-head comparison of ASCs and SVF for the treatment of knee OA in humans. ASC and SVF injections both substantially improved knee pain and function at all follow-up time points, although ASC injections demonstrated significantly better improvements with regard to the MCID and PASS for the pain VAS and the MCID for the KOOS5 at 12 months. There appears to be no benefit to a booster ASC injection after initial treatment. Given less donor-site morbidity and equivalent superior outcomes at 2 years, the use of ASCs over SVF in the treatment of knee OA may be warranted.

Factors affecting cell viability and the yield of adipose-derived stromal vascular fraction

The stromal vascular fraction (SVF) is isolated from adipose tissue and has tremendous regenerative potential for proliferation and differentiation. This study aimed to investigate the factors affecting the cell yield and viability of the SVF to improve the outcomes of its clinical applications and enhance its clinical usage. We performed a retrospective analysis with 121 patients who underwent liposuction to harvest adipose-derived SVF. We recorded patient demographic and clinical characteristics, including age, sex, body mass index (BMI), blood type, medical comorbidities, and smoking and alcohol consumption. As for operative variables, we noted the amount of lipoaspirate and the donor areas, including the lower and entire abdomen. The viability and the cell count of SVF were documented. Sex was a statistically significant factor for viability rate (p < 0.015) and cell count (p < 0.009). Men had higher viability, while women had higher cell counts. We found a statistically significant difference in the presence of hypertension (p = 0.024) and alcohol consumption (p = 0.024). There was a statistically significant relationship between cell count and age (p < 0.001), BMI (p = 0.006), and amount of lipoaspirate (p < 0.001). Sex had significant associations with cell count and viability, while age, BMI, and lipoaspirate amount were significantly associated with cell count. Hypertension and alcohol consumption significantly affected cell count, which is the first such report of this association. Surgeons could apply this knowledge to patient selection for optimal treatment outcomes. Additionally, understanding these factors can help manage patient expectations.

Paracrine effect of the stromal vascular fraction containing M2 macrophages on human chondrocytes through the Smad2/3 signaling pathway.

The adipose-derived stromal vascular fraction (SVF) is composed of a heterogeneous mix of adipose-derived stem cells (ADSCs), macrophages, pericytes, fibroblasts, blood, and other cells. Previous studies have found that the paracrine effects of SVF cells may be therapeutic, but their role in osteoarthritis treatment remains unclear. This study aimed to investigate the therapeutic effect of SVF cells on chondrocytes. Chondrocytes were seeded on culture plates alone (control) or cocultured with SVF or ADSCs on cell culture inserts. After 48 h of coculture, chondrocyte collagen II, tissue inhibitors of metalloproteinases-3 (TIMP-3), and matrix metalloproteinases-13 (MMP-13) messenger RNA (mRNA) expression levels were evaluated using reverse-transcription polymerase chain reaction, and the transforming growth factor-β (TGF-β) levels in the supernatant were measured using ELISA. Immunohistochemical staining and flow cytometry were used to evaluate the macrophages in the SVF. These macrophages were characterized according to phenotype using the F4/80, CD86, and CD163 markers. To determine whether the Smad2/3 signaling pathways were involved, the chondrocytes were pre-treated with a Smad2/3 phosphorylation inhibitor and stimulated with the SVF, and then Smad2/3 phosphorylation levels were analyzed using western blot. The mRNA expression levels of various paracrine factors and chondrocyte pellet size were also assessed. Collagen II and TIMP-3 expression were higher in the SVF group than in the ADSC group and controls, while MMP-13 expression was the highest in the ADSC group and the lowest in the controls. TGF-β levels in the SVF group were also elevated. Immunohistochemical staining and flow cytometry revealed that the macrophages in the SVF were of the anti-inflammatory phenotype. Western blot analysis showed that the SVF increased Smad2/3 phosphorylation, while Smad2/3 inhibitors decreased phosphorylation. Smad2/3 inhibitors also reduced the expression of various other paracrine factors and decreased chondrocyte pellet size. These findings suggested that the paracrine effect of heterogeneous cells, such as anti-inflammatory macrophages, in the SVF partly supports chondrocyte regeneration through TGF-β-induced Smad2/3 phosphorylation.

Abstract 6147: Immune population changes in mammary adipose tissue during the development of obesity and their influence on triple negative breast cancer progression

Abstract Obesity is associated with increased risk for many cancer types, including triple-negative breast cancer (TNBC). In addition to obesity’s role in TNBC pathogenesis, it is also recognized as a marker of poor prognosis for survival in pre- and post-menopausal women. However, the mechanism underlying how obesity leads to worsened TNBC progression remains unclear. In this study, we aim to characterize the immune population changes in mammary adipose during the development of obesity and their impact on TNBC progression. First, we utilized a diet-induced obesity mouse model to evaluate tumor growth under obese condition. Four weeks after E0771 (TNBC cell line) mammary fat pad injection, TNBC tumors volume were 1.6-fold larger in obese C57BL/6 mice compared to their lean counterparts. To profile the transcriptome changes induced by obesity, we performed single-cell RNA-Seq on mammary adipose stromal vascular fraction and TNBC allograft from lean and obese C57BL/6 female mice. In TNBC allografts, obesity upregulated genes enriched in the extracellular matrix and epithelial-mesenchymal transition pathways in cancer cells, creating a more aggressive phenotype. Consistent with the heavier TNBC tumor burden, lymphocyte infiltration decreased while myeloid population increased in TNBC allografts from obese mice, which indicated an immunosuppressed tumor microenvironment. Macrophage was the dominant population among Cd45+ cells in tumors, constituting 41.2% in lean and 51.2% in obese mice. An increase of adipose tissue macrophage (ATM) population (2.2-fold increase in obese vs lean adipose; 5.6-fold increase in tumor-bearing vs control adipose) was also observed in the obese or tumor-bearing mammary adipose stroma. Using Gene Set Enrichment Analysis (GSEA), we found that ATM adapted to the obese condition by upregulating fatty acid metabolism and adipogenesis hallmarks while downregulating IFNγ response, allograft rejection, and TNFα signal hallmarks. When encountering TNBC development, ATM downregulated similar proinflammatory pathways, while upregulated the hypoxia and glycolysis pathways. Through integrative analysis, we found that the complement pathway is the primary pathway upregulated in ATM under both obese and tumor growth conditions. Major components of the complement pathway, C1qa and C1qb, were significantly upregulated in ATM by TNBC and obesity. Interestingly, C1qa and C1qb were also upregulated in bone-marrow derived macrophage during M2 polarization and when co-culturing with E0771. Taken together, our data showed that the C1qahi/C1qbhi macrophage expanded in mammary adipose tissue during the development of obesity and TNBC, which created an immunosuppressed microenvironment to facilitate TNBC progression. This suggests that the complement pathway may serve as a potential immune therapy target for obese TNBC patients. Citation Format: Shimeng Liu, Yi Zhang, Myles Brown. Immune population changes in mammary adipose tissue during the development of obesity and their influence on triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6147.

Viewing stromal vascular fraction de novo vessel formation and association with host microvasculature using the rat mesentery culture model.

The objective of the study is to demonstrate the innovation and utility of mesenteric tissue culture for discovering the microvascular growth dynamics associated with adipose-derived stromal vascular fraction (SVF) transplantation. Understanding how SVF cells contribute to de novo vessel growth (i.e., neovascularization) and host network angiogenesis motivates the need to make observations at single-cell and network levels within a tissue. Stromal vascular fraction was isolated from the inguinal adipose of adult male Wistar rats, labeled with DiI, and seeded onto adult Wistar rat mesentery tissues. Tissues were then cultured in MEM +10% FBS for 3 days and labeled for BSI-lectin to identify vessels. Alternatively, SVF and tissues from green fluorescent-positive (GFP) Sprague Dawley rats were used to track SVF derived versus host vasculature. Stromal vascular fraction-treated tissues displayed a dramatically increased vascularized area compared to untreated tissues. DiI and GFP+ tracking of SVF identified neovascularization involving initial segment formation, radial outgrowth from central hub-like structures, and connection of segments. Neovascularization was also supported by the formation of segments in previously avascular areas. New segments characteristic of SVF neovessels contained endothelial cells and pericytes. Additionally, a subset of SVF cells displayed the ability to associate with host vessels and the presence of SVF increased host network angiogenesis. The results showcase the use of the rat mesentery culture model as a novel tool for elucidating SVF cell transplant dynamics and highlight the impact of model selection for visualization.

Adipose Stromal Vascular Fraction Reverses Mitochondrial Hyperfission in Coronary Microvessels in Aging

IntroductionIn aging post‐menopausal women, Coronary Microvascular Disease (CMD) leads to hyperconstricted tone, reduced perfusion and chronic micro‐ischemia with angina. We previously showed an age‐related increase in coronary microvascular ROS alongside increased prooxidant gene and protein expression associated with blunted vasodilation. Adipose Stromal Vascular Fraction (SVF) is a heterogenous cell population that reduces vascular ROS to improve vasodilation. Oxidative stress with aging may be mediated by mitochondrial dysfunction, including fission/fusion imbalance. Therefore, we hypothesize aging leads to mitochondrial hyperfission and reduced fusion gene and protein expression, reversed by SVF therapy.MethodsCoronary microvessels from young (YC, 4 months), old (OC, 24 months), or old + SVF (OSVF, tail vein injection 4 weeks prior to sacrifice) from female Fischer 344 rats were assessed via RNA sequencing (n=3) and immunofluorescence imaging of fission/fusion proteins Dynamin Related Protein 1 (DRP‐1) and Mitofusin 1 &amp; 2 (MFN1 &amp; 2). Morphometric analysis was accomplished using ImageJ and the MiNA Image‐J tool to analyze 60x confocal images of microvessels stained with antibodies against mitochondrial TOM20. Statistics were preformed using One‐Way ANOVA with Bonferroni post hoc analysis.ResultsGene expression of DRP‐1 was significantly reduced, and MFN‐1 significantly enhanced in OSVF vs. OC (p = 0.035, 0.019). There was no significant difference in gene expression of fission or fusion mediators Fis‐1, MFN‐2 or Opa‐1. Protein expression of DRP‐1 was enhanced with aging, reduced to youthful levels by SVF (p = 0.007, 0.033) (Fig.1a). There were no differences in MFN1 or MFN2 protein expression with aging, however, protein expression of MFN‐1 was significantly increased after SVF (YC vs. OSVF p = 0.003, OC vs. OSVF p &lt; .001). Mitochondrial aspect ratio and form factor were reduced in aging (p = 0.024, &lt; 0.001), restored to youthful levels by SVF (p = &lt; 0.001, &lt; 0.001) (Fig. 1b). Mitochondrial fission factor was elevated with aging (p = &lt; 0.001), reversed by SVF therapy (p = &lt; 0.001) (Fig. 1c). Mitochondrial area was reduced with aging (p = 0.002). Mitochondrial branch length and numbers of branches within a network were greatest in youth, significantly attenuated with aging (p &lt; 0.001, &lt; 0.001), and significantly increased (albeit not to youthful levels) with SVF therapy (OC vs. OSVF: p = 0.017, 0.011, YC vs. OSVF: p = 0.004, 0.003) (Fig. 1d).ConclusionsDuring youth, mitochondrial morphology is more rod‐like and interconnected, vs. punctate and isolated in aging. Injection of SVF reversed mitochondrial circularity and partially restored mitochondrial network density in aging. These findings were complimented by enhanced expression of DRP‐1 with aging with reduced DRP‐1 and enhanced MFN‐1 expression with SVF. SVF is a plausible therapy for CMD by reversing mitochondrial dysfunction including alleviating oxidative stress, attenuating mitochondrial fission, and rejuvenating mitochondrial fusion.

Heterogeneity of adipose stromal vascular fraction cells from the different harvesting sites in rats.

In both veterinary and human health, regenerative medicine offers a promising cure for various disorders. One of the rate-limiting challenges in regenerative medicine is the considerable time and technique required to expand and grow cells in culture. Therefore, the stromal vascular fraction (SVF) shows a significant promise for various cell therapy approaches. The present study aimed to define and investigate the optimal harvest site of freshly isolated SVF cells from various adipose tissue (AT) depot sites in the female Sprague-Dawley (S.D.) rat. First, hematoxylin and eosin (H&E) were used to analyze the morphological variations in AT samples from peri-ovarian, peri-renal, mesenteric, and omental sites. The presence of putative stromal cells positive CD34 was detected using immunohistochemistry. Then, the isolated SVF cells were examined for cell viability and cellular yield differences. Finally, the expression of mesenchymal stem cells and hematopoietic markers in the SVF cells subpopulation was studied using flow cytometry. The pluripotent gene expression profile was also evaluated. CD34 staining of the omental AT was substantially higher than those of other anatomical sites. Despite having the least quantity of fat, omental AT has the highest SVF cell fraction and viable cells. Along with CD90 and CD44 higher expression, Oct4, Sox2, and Rex-1 genes levels were higher in SVF cells isolated from the omental AT. To conclude, omental fat is the best candidate for SVF cell isolation in female S.D. rats with the highest SVF cell fraction with higher MSCs phenotypes and pluripotency gene expression.

Advancing Regenerative Cellular Therapies in Non-Scarring Alopecia

Alopecia or baldness is a common diagnosis in clinical practice. Alopecia can be scarring or non-scarring, diffuse or patchy. The most prevalent type of alopecia is non-scarring alopecia, with the majority of cases being androgenetic alopecia (AGA) or alopecia areata (AA). AGA is traditionally treated with minoxidil and finasteride, while AA is treated with immune modulators; however, both treatments have significant downsides. These drawbacks compel us to explore regenerative therapies that are relatively devoid of adverse effects. A thorough literature review was conducted to explore the existing proven and experimental regenerative treatment modalities in non-scarring alopecia. Multiple treatment options compelled us to classify them into growth factor-rich and stem cell-rich. The growth factor-rich group included platelet-rich plasma, stem cell-conditioned medium, exosomes and placental extract whereas adult stem cells (adipose-derived stem cell-nano fat and stromal vascular fraction; bone marrow stem cell and hair follicle stem cells) and perinatal stem cells (umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), Wharton jelly-derived MSCs (WJ-MSCs), amniotic fluid-derived MSCs (AF-MSCs), and placental MSCs) were grouped into the stem cell-rich group. Because of its regenerative and proliferative capabilities, MSC lies at the heart of regenerative cellular treatment for hair restoration. A literature review revealed that both adult and perinatal MSCs are successful as a mesotherapy for hair regrowth. However, there is a lack of standardization in terms of preparation, dose, and route of administration. To better understand the source and mode of action of regenerative cellular therapies in hair restoration, we have proposed the “À La Mode Classification”. In addition, available evidence-based cellular treatments for hair regrowth have been thoroughly described.

Effect of uncultured adipose-derived stromal vascular fraction on preventing urethral stricture formation in rats

Urethral stricture (US) remains a challenging disease without effective treatment options due to the high recurrence rate. This study aims to evaluate the preventive effect of uncultured adipose derived stromal vascular fraction (SVF) on urethral fibrosis in a rat model of US. Results demonstrated that US rats displayed hyperechogenic urethral wall with a narrowed lumen compared with sham rats, while SVF rats exhibited less extensive urethral changes. By histology, US rats showed obvious submucosal fibrosis in the urethral specimens, while SVF rats exhibited mild submucosal fibrosis with less extensive tissue changes. Furthermore, US rats showed increased gene and protein expression of collagen I (2.0 ± 0.2, 2.2 ± 0.2, all were normalized against GAPDH, including the following), collagen III (2.5 ± 0.3, 1.2 ± 0.1), and TGFβ1R (2.8 ± 0.3, 1.9 ± 0.2), while SVF cells administration contributed to decreased gene and protein expression of collagen I (1.6 ± 0.2, 1.6 ± 0.2), collagen III (1.8 ± 0.4, 0.9 ± 0.1), and TGFβ1R (1.8 ± 0.3, 1.3 ± 0.2), in parallel with the improvement of vascularization and increased expression of VEGF (1.7 ± 0.1) and bFGF (3.1 ± 0.3). Additionally, SVF served anti-inflammatory effect through regulation of inflammatory cytokines and cells, accompanied with conversion of the macrophage phenotype. Our findings suggested that uncultured SVF presented an inhibitory effect on stricture formation at an early stage of urethral fibrosis.

An In Vitro Study of the Effects of Mechanical and Enzymatic Isolation of Stromal Vascular Fraction on Wound Healing.

The adipose-derived stromal vascular fraction (SVF) is considered to be an attractive source of stem cells in cell therapy. Besides stem cells, it also contains functional cells, such as macrophages, precursor cells, somatic stem cells, and pericytes. Collagenase digestion is the most frequently used method to isolate SVF, but it is time-consuming and costly and has some problems, such as infectious agents and immune reactions. In this research, we compared the yield, cell population ratios, and cell viability when isolating SVF by the ultrasonic physics (U-SVF) method and traditional enzymatic method (E-SVF). Then, we isolated exosomes from U-SVF and E-SVF, respectively, and cocultured them with fibroblasts to investigate the potential of applying this cell secretion in wound repair. The results showed that there was no significant difference between the ultrasonic method and enzymatic method in terms of cell viability, cell numbers, or the expression of CD markers of stem cells. However, exosome analysis identified a greater number and smaller size of exosome particles obtained by U-SVF. In terms of cell proliferation efficiency, although the proliferation efficiency of U-SVF was lower than that of E-SVF. Trilineage differentiation experiments revealed that both E-SVF and U-SVF had good differentiation ability, owing to high stem cell content. Finally, E-SVF and U-SVF exosomes were cocultured with fibroblasts. The efficiency of fibroblast migration increased in the SVF exosome treated groups, and the expression of related genes (integrin α5β1) was slightly upregulated; however, the expression of FAK, AKT, ERK, and RhoA was significantly upregulated at 24 hours. From the abovementioned experiments, we found that there was no significant difference in stem cell-related characteristics between SVF isolated by ultrasonic cavitation and SVF isolated by the enzymatic method. In addition, exosomes secreted by SVF may have excellent therapeutic effect on skin injuries, which provides a new viewpoint and therapeutic strategy for soft tissue repair.

Osteogenic Induction with Silicon Hydroxyapatite Using Modified Autologous Adipose Tissue-Derived Stromal Vascular Fraction: In Vitro and Qualitative Histomorphometric Analysis.

Large bone defects requiring invasive surgical procedures have long been a problem for orthopedic surgeons. Despite the use of autologous bone grafting, satisfactory results are often not achieved due to associated limitations. Biomaterials are viable alternatives and have lately been used in association with Stromal Vascular Fraction (SVF), stem cells, and signaling factors for bone tissue engineering (BTE). The objective of the current study was to assess the biocompatibility of Silicon Hydroxyapatite (Si-HA) and to improve osteogenic potential by using autologous adipose-derived SVF with Si-HA in a rabbit bone defect model. Si-HA granules synthesized using a wet precipitation method were used. They were characterized using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD). A hemolysis assay was used to assess the hemolytic effects of Si-HA, while cell viability was assessed through Alamar Blue assay using MC3T3 mouse osteoblasts. The osteogenic potential of Si-HA both alone and with enzymatically/non-enzymatically-derived SVF (modified) was performed by implantation in a rabbit tibia model followed by histomorphometric analysis and SEM of dissected bone after six weeks. The results showed that Si-HA granules were microporous and phase pure and that the addition of Silicon did not influence Si-HA phase composition. Si-HA granules were found to be non-hemolytic on the hemolysis assay and non-toxic to MC3T3 mouse osteoblasts on the Alamar Blue assay. Six weeks following implantation Si-HA showed high biocompatibility, with increased bone formation in all groups compared to control. Histologically more mature bone was formed in the Si-HA implanted along with non-enzymatically-derived modified SVF. Bone formation was observed on and around Si-HA, reflecting osseointegration. In conclusion, Si-HA is osteoconductive and promotes osteogenesis, and its use with SVF enhances osteogenesis.

Therapeutic effect of adipose stromal vascular fraction spheroids for partial bladder outlet obstruction induced underactive bladder

BackgroundUnderactive bladder (UAB) is a common clinical problem but related research is rarely explored. As there are currently no effective therapies, the administration of adipose stromal vascular fraction (ad-SVF) provides a new potential method to treat underactive bladder.MethodsMale Sprague–Dawley rats were induced by partial bladder outlet obstruction (PBOO) for four weeks and randomly divided into three groups: rats treated with PBS (Sham group); rats administrated with ad-SVF (ad-SVF group) and rats performed with ad-SVF spheroids (ad-SVFsp group). After four weeks, urodynamic studies were performed to evaluate bladder functions and all rats were sacrificed for further studies.ResultsWe observed that the bladder functions and symptoms of UAB were significantly improved in the ad-SVFsp group than that in the Sham group and ad-SVF group. Meanwhile, our data showed that ad-SVF spheroids could remarkably promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation in bladder tissue than that in the other two groups. Moreover, ad-SVF spheroids increased the expression levels of bFGF, HGF and VEGF-A than ad-SVF. IVIS Spectrum small-animal in vivo imaging system revealed that ad-SVF spheroids could increase the retention rate of transplanted cells in bladder tissue.ConclusionsAd-SVF spheroids improved functions and symptoms of bladder induced by PBOO, which contributes to promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation. Ad-SVF spheroids provide a potential treatment for the future patients with UAB.

The Effect of Autologous Adipose-Derived Stromal Vascular Fractions on Cartilage Regeneration Was Quantitatively Evaluated Based on the 3D-FS-SPGR Sequence: A Clinical Trial Study.

Background Numerous reports confirmed the safety and clinical efficacy of autologous adipose-derived stromal vascular fractions (SVF), which have recently been used to treat osteoarthritis (OA). However, there is still no consensus as to whether SVF can promote cartilage regeneration. Herein, the purpose of our study was to evaluate the effectiveness of SVF versus hyaluronic acid (HA) in cartilage regeneration by establishing a cartilage model based on the three-dimensional fat-suppressed spoiled gradient recalled echo (3D-FS-SPGR) sequence. Methods Patients with symptomatic OA were recruited in our research, who were randomized into two groups. Meanwhile, patients in Kellgren-Lawrence (K-L) grades 2 and 3 were distinguished in each group. In the test group, patients received SVF injections of the knee, while patients in the control group received the same dose of HA. Each patient underwent the 3D-FS-SPGR sequence to establish a cartilage model at baseline, 6 months, and 12 months, respectively. The cartilage was characterized into six regions, and relevant parameters of the cartilage model were counted. Clinical and radiographic scores were recorded in one-year follow-up. Results In all regions, the thickness and volume of cartilage defect and the volume of healthy cartilage were improved to some extent in the test group, especially the medial femoral condyle (MF) and medial tibial condyle (MT). In grades 2 and 3, the thickness and volume of cartilage defect decreased by 0.92 ± 0.18 mm and 1.03 ± 0.23 mm and 84.00 ± 32.30 mm3 and 130.30 ± 49.56 mm3 in MF and by 0.96 ± 0.22 mm and 0.99 ± 0.14 mm and 64.18 ± 21.40 mm3 and 95.11 ± 19.93 mm3 in MT, respectively. No such phenomenon was observed in the control group. Meanwhile, the SVF-treated knees showed significant improvement in clinical and radiographic scores at 12 months. Nevertheless, these scores of the control group became worse at 12-month follow-up visit. Conclusion Taken together, this study shows that intra-articular injection of SVF markedly improved the clinical symptoms without adverse events, thereby repairing the damaged articular cartilage through cartilage regeneration.