Viability Of Adipose Stem Cells Research Articles

The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic differentiation of adipose stem cells

Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteogenic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia, and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g. Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition. Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 % oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that intermittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone tissue engineering approaches.

Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells.

To achieve the full therapeutic potential of implanted adipose stem cells (ASCs) in vivo, it is crucial to improve the viability and pro-angiogenic properties of the stem cells. Here, we first simulated the conditions of ischemia and hypoxia using the in vitro oxygen-glucose deprivation (OGD) model and confirmed that hypoxic preconditioning of ASCs could provide improved protection against OGD and enhance ASC viability. Second, we assessed the effect of hypoxic preconditioning on pro-angiogenic potential of ASCs, with a particular focus on the role of vascular endothelial growth factor-A (VEGF-A) and stromal derived factor-1a (SDF-1a) paracrine activity in mediating angiogenesis. We found that the conditioned medium of ASCs (ASCCM) with hypoxic preconditioning enhanced angiogenesis by a series of angiogenesis assay models in vivo and in vitro through the upregulation of and a synergistic effect between VEGF-A and SDF-1a. Finally, to investigate the possible downstream mechanisms of VEGF/VEGFR2 and SDF-1a/CXCR4 axes-driven angiogenesis, we evaluated relevant protein kinases involved the signal transduction pathway of angiogenesis and showed that VEGF/VEGFR2 and SDF-1a/CXCR4 axes may synergistically promote angiogenesis by activating Akt. Collectively, our findings demonstrate that hypoxic preconditioning may constitute a promising strategy to enhance cellular viability and angiogenesis of transplanted ASCs, therein improving the success rate of stem cell-based therapies in tissue engineering.

Blood Impairs Viability of Fat Grafts and Adipose Stem Cells: Importance of Washing in Fat Processing.

Fat processing plays a pivotal role in graft survival. Each component of the blood in lipoaspirate affects fat survival in different ways, but the mechanisms are not clear. The aim of this study was to investigate, by various experimental methods, the effect of blood on the viability of fat grafts and adipose stem cells (ASCs). Blood and fat samples were obtained from 6 female patients undergoing aesthetic liposuction. For the in vivo experiment, we compared fat mixed with normal saline or various ratios of blood in nude mice. The samples were explanted at 2 and 8 weeks to evaluate the gross volume retention and histologic and immunohistochemical characteristics. For in vitro experiments, ASCs were pretreated with hemoglobin at different concentrations and for different times. We then assessed the proliferation, migration, adipogenesis, and reactive oxygen species production of ASCs. Blood in the graft led to a decrease in graft viability, as evaluated by general observation and histologic and immunohistochemical morphology in vivo. In vitro experiments showed inhibited proliferation, migration, and adipogenesis, and increased reactive oxygen species production in ACSs, after hemoglobin treatment, suggesting impaired ASC viability. This study suggests that blood impairs the viability of fat grafts and ASCs and provides evidence that washing to remove blood is important in fat processing.

Bioactive borate glass triggers phenotypic changes in adipose stem cells.

A bioactive borate glass, 13-93B3 (B3), has been used successfully in the clinic to treat chronic, nonhealing wounds without scarring. However, the mechanism by which B3 stimulates wound healing is poorly understood. Because adipose stem cells (ASCs) have been shown to have multiple roles in wound repair, we hypothesized that B3 triggers ASCs. In this study, we evaluate the effects of B3 on ASC survival, migration, differentiation, and protein secretion in vitro. In concentrations ≤10 mg/ml, B3 did not affect ASC viability under static conditions. B3 promoted the migration of ASCs but did not increase differentiation into bone or fat. B3 also decreased ASCs secretion of collagen I, PAI-1, MCP-1, DR6, DKK-1, angiogenin, IL-1, IGFBP-6, VEGF, and TIMP-2; increased expression of IL-1R and E-selectin; had a transient decrease in IL-6 secretion; and had a transient increase in bFGF secretion. Together, these results show that B3 alters the protein secretion of ASCs.

Application of honey as a protective material in maintaining the viability of adipose stem cells in burn wound healing: A histological, molecular and biochemical study

Enhanced resistance to oxidative stress makes the adipose stem cells (ASCs) able to promote wound repair and regeneration. Such cells can be achieved by addition of an anti-oxidant to cell culture medium. In this study, a combination of honey (H) and ASCs was applied on burn wounds and the injured area was then covered by a tegaderm (T) dressing in a rat model. Wound healing was evaluated by histopathological, histomorphometrical, molecular, scanning electron microscopy, and biochemical assessments on days 7, 14, and 28 post-wounding. Treatment with ASCs-containing honey (T-H/ASC) resulted in substantial reduction in the level of pro-inflammatory cytokines including interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) in wound bed, at 7 day post-surgery. T-H/ASCs also elevated the level of bFGF expression, indicating that ASCs enhanced angiogenesis, at 7 day post-wounding. T-H/ASCs significantly improved angiogenesis, re-epithelialization, and granulation tissue formation compared to other treatment regimes, at 14 day post-surgery. These outcomes were in exceptionally good agreement with the histological and biochemical findings. Increased bFGF level in the T-H/ASCs treated wounds at the 28th day post-surgery showed the anti-scarring activity of ASCs. Honey can be considered as a protective material in maintaining the viability of ASCs and improving the cellular resistance to oxidative stress. Furthermore, combination of ASCs and honey can provide a nutrient media for the ASCs and enhance the ability of regeneration of the ASC-based therapies.

Current and Future Trends in Adipose Stem Cell Differentiation into Neuroglia.

Neurological diseases and disorders pose a challenge for treatment and rehabilitation due to the limited capacity of the nervous system to repair itself. Adipose stem cells (ASCs) are more pliable than any adult stem cells and are capable of differentiating into non-mesodermal tissues, including neurons. Transdifferentiating ASCs to specific neuronal lineage cells enables us to deliver the right type of cells required for a replacement therapy into the nervous system. Several methodologies are being explored and tested to differentiate ASCs to functional neurons and glia with cellular factors and chemical compounds. However, none of these processes and prototypes has been wholly successful in changing the cellular structure and functional status of ASCs to become identical to neuroglial cells. In addition, successful integration and functional competence of these cells for use in clinical applications remain problematic. Photobiomodulation or low-level laser irradiation has been successfully applied to not only improve ASC viability and proliferation but has also shown promise as a possible enhancer of ASC differentiation. Studies have shown that photobiomodulation improves the use of stem cell transplantation for neurological applications. This review investigates current neuro-differentiation inducers and suitable methodologies, including photobiomodulation, utilizing ASCs for induction of differentiation into neuronal lineages.

3D bioprinting of stem cells and polymer/bioactive glass composite scaffolds for bone tissue engineering.

A major limitation of using synthetic scaffolds in tissue engineering applications is insufficient angiogenesis in scaffold interior. Bioactive borate glasses have been shown to promote angiogenesis. There is a need to investigate the biofabrication of polymer composites by incorporating borate glass to increase the angiogenic capacity of the fabricated scaffolds. In this study, we investigated the bioprinting of human adipose stem cells (ASCs) with a polycaprolactone (PCL)/bioactive borate glass composite. Borate glass at the concentration of 10 to 50 weight %, was added to a mixture of PCL and organic solvent to make an extrudable paste. ASCs suspended in Matrigel were ejected as droplets using a second syringe. Scaffolds measuring 10 x 10 x 1 mm3 in overall dimensions with pore sizes ranging from 100 - 300 μm were fabricated. Degradation of the scaffolds in cell culture medium showed a controlled release of bioactive glass for up to two weeks. The viability of ASCs printed on the scaffold was investigated during the same time period. This 3D bioprinting method shows a high potential to create a bioactive, highly angiogenic three-dimensional environment required for complex and dynamic interactions that govern the cell’s behavior in vivo.

Electrically Stimulated Adipose Stem Cells on Polypyrrole-Coated Scaffolds for Smooth Muscle Tissue Engineering.

We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1ms) or short pulse (0.25ms) biphasic electric current at a frequency of 10Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.

A Simple Method to Determine the Purity of Adipose-Derived Stem Cell-Based Cell Therapies.

In order to provide direction to academia, industry, and regulatory authorities regarding purity assessment for adipose tissue-derived cells, this report describes a simple tool to facilitate development of international standards based on reproducible parameters and endpoints that may systematize cellular products across boundaries and accelerate the delivery of safe and effective adipose stem cell (ASC)-based tools to the medical community and the patients it serves. This tool (a) can discriminate specifically fibroblasts and ASCs and (b) can be rapidly implemented and performed before the release of the ASC-based therapy (a response within 24 hours).

Effect of liraglutide on proliferation and differentiation of human adipose stem cells

Glucagon-Like Peptide-1 (GLP-1) receptor agonists, used as glucose-lowering drugs, also induce weight loss by inhibiting food intake. The present study was aimed at the assessment of the in vitro effects of the GLP-1 receptor agonist liraglutide on proliferation and differentiation of human adipose stem cells (ASC) obtained from subcutaneous adipose tissue of morbidly obese subjects undergoing bariatric surgery.Liraglutide (10–100 nM) significantly inhibited ASC proliferation and viability, with a maximum effect at 6 days of culture (45% and 50%, for liraglutide 10 and 100 nM, respectively); the effect was reverted by exendin 9–39. Glucose uptake was significantly reduced by liraglutide in a dose dependent manner. Treatment with liraglutide reduced intracellular lipid accumulation in differentiating ASC, together with FABP-4 mRNA expression (−18%, −23%, −46%, for 1 nM, 10 nM and 100 nM, respectively), whereas it stimulated adiponectin (APN) expression (1.86-, 2.64-, 2.28-fold increase, for 1 nM, 10 nM and 100 nM, respectively).Liraglutide exerts effects on human adipose cell precursors, inhibiting proliferation and differentiation, while stimulating the expression of the insulin-sensitizing adipokine APN. These effects could contribute to the actions of GLP-1 receptor agonists on body weight and insulin sensitivity.

Effects of chitosan and bioactive glass modifications of knitted and rolled polylactide-based 96/4 L/D scaffolds on chondrogenic differentiation of adipose stem cells

The performance of biodegradable knitted and rolled 3-dimensional (3D) polylactide-based 96/4 scaffolds modified with bioactive glass (BaG) 13-93, chitosan and both was compared with regard to the viability, proliferation and chondrogenic differentiation of rabbit adipose stem cells (ASCs). Scaffold porosities were determined by micro-computed tomography (μCT). Water absorption and degradation of scaffolds were studied during 28-day hydrolysis in Tris-buffer. Viability, number and differentiation of ASCs in PLA96/4 scaffolds were examined in vitro. The dimensions of the scaffolds were maintained during hydrolysis and mass loss was detected only in the BaG13-93 containing scaffolds. ASCs adhered and proliferated on each scaffold type. Cell aggregation and expression of chondral matrix components improved in all scaffold types in chondrogenic medium. Signs of hypertrophy were detected in the modified scaffolds but not in the plain PLA96/4 scaffold. Chondrogenic differentiation was most enhanced in the presence of chitosan. These findings indicate that the plain P scaffold provided a good 3D-matrix for ASC proliferation whereas the addition of chitosan to the PLA96/4 scaffold induced chondrogenic differentiation independent of the medium. Accordingly, a PLA96/4 scaffold modified by chitosan could provide a functional and bioactive basis for tissue-engineered chondral implants.

Growth and Osteogenic Differentiation of Adipose Stem Cells on PLA/Bioactive Glass and PLA/β-TCP Scaffolds

The aim of this study was to compare the effects of novel three-dimensional composite scaffolds consisting of a bioactive phase (bioactive glass or beta-tricalcium phosphate [beta-TCP] 10 and 20 wt%) incorporated within a polylactic acid (PLA) matrix on viability, distribution, proliferation, and osteogenic differentiation of human adipose stem cells (ASCs). The viability and distribution of ASCs on the bioactive composite scaffolds was evaluated using Live/Dead fluorescence staining, environmental scanning electron microscopy, and scanning electron microscopy. There were no differences between the two concentrations of bioactive glass and beta-TCP in PLA scaffolds on proliferation and osteogenic differentiation of ASCs. After 2 weeks of culture, DNA content and alkaline phosphatase (ALP) activity of ASCs cultured on PLA/beta-TCP composite scaffolds were higher relative to other scaffold types. Interestingly, the cell number was significantly lower, but the relative ALP/DNA ratio of ASCs was significantly higher in PLA/bioactive glass scaffolds than in other three scaffold types. These results indicate that the PLA/beta-TCP composite scaffolds significantly enhance ASC proliferation and total ALP activity compared to other scaffold types. This supports the potential future use of PLA/beta-TCP composites as effective scaffolds for tissue engineering and as bone replacement materials.