The Relationship of a Combination of Human Adipose Tissue-Derived Stem Cells and Frozen Fat with the Survival Rate of Transplanted Fat

BACKGROUND: The survival rate of grafted fat is difficult to predict, and repeated procedures are frequently required. In this study, the effects of the freezing period of harvested adipose tissue and the addition of human adipose tissue-derived stem cells (ASCs) on the process of fat absorption were studied. METHODS: Adipose tissue was obtained from patients who underwent a lipoaspirated fat graft. The fat tissue was cryopreserved at -20degrees C in a domestic refrigerator. A total of 40 nude mice were used. The mice in the experimental group received three different subcutaneous injections in the back: an injection of fresh fat and ASCs, an injection of fat that had been frozen for one month and ASCs, and an injection of fat that had been frozen for two months and ASCs. The control mice received fat grafts without ASCs. The mice were sacrificed at four or eight weeks after the procedure, and the grafted fat tissues were harvested. The extracted fat was evaluated using photographic analysis, volume measurements, and histological examination. RESULTS: In the control group, the fat resorption rates four weeks after transplantation in the grafts of fresh fat, fat that had been frozen for one month, and fat that had been frozen for two months were 21.14%, 22.46%, and 42.56%, respectively. In the experimental group, the corresponding resorption rates were 6.68%, 13.0%, and 33.9%, respectively. CONCLUSIONS: ASCs can increase the fat graft survival rate. The use of ASCs in fat grafting can reduce the need for repeated fat grafts and provide good long term results.

Effects of Platelet-Rich Plasma, Adipose-Derived Stem Cells, and Stromal Vascular Fraction on the Survival of Human Transplanted Adipose Tissue

Traditional adipose tissue transplantation has unpredictable viability and poor absorption rates. Recent studies have reported that treatment with platelet-rich plasma (PRP), adipose-derived stem cells (ASCs), and stromal vascular fraction (SVF) are related to increased survival of grafted adipose tissue. This study was the first simultaneous comparison of graft survival in combination with PRP, ASCs, and SVF. Adipose tissues were mixed with each other, injected subcutaneously into the back of nude mice, and evaluated at 4, 8, and 12 weeks. Human adipocytes were grossly maintained in the ASCs and SVF mixtures. Survival of the adipose tissues with PRP was observed at 4 weeks and with SVF at 8 and 12 weeks. At 12 weeks, volume reduction in the ASCs and SVF mixtures were 36.9% and 32.1%, respectively, which were significantly different from that of the control group without adjuvant treatment, 51.0%. Neovascular structures were rarely observed in any of the groups. Our results suggest that the technique of adding ASCs or SVF to transplanted adipose tissue might be more effective than the conventional grafting method. An autologous adipose tissue graft in combination with ASCs or SVF may potentially contribute to stabilization of engraftment.

Morphine Attenuates Endothelial Cell Adhesion Molecules Induced by the Supernatant of LPS-Stimulated Colon Cancer Cells

A large reservoir of bacterial lipopolysaccharide (LPS) is available in the colon and this could promote colon cancer metastasis by enhancing tumor cell adhesion, intravasation, and extravasation. Furthermore, adhesion molecules like ICAM-1, VCAM-1, and E-selectin play important roles in the adhesion of tumor cells to endothelium. This study was designed to determine whether morphine can attenuate the expressions of adhesion molecules up-regulated by the supernatant of LPS-stimulated HCT 116 colon cancer cells (LPS-Sup). In this study, we divided to three groups by cell-growth medium of human umbilical vascular endothelial cells (HUVECs): the control group was incubated in growth factor-free endothelial medium, the Sup group was incubated in the supernatant of HCT 116 cells (Sup), and the LPS-Sup group was incubated in LPS-Sup. To observe effect of morphine to the adhesion molecules expressions in the LPS-Sup group, we co-treated morphine with LPS or added it to LPS-Sup. Adhesion molecule expressions on HUVECs in all three groups were measured during incubation period. Consquentially, ICAM-1, VCAM-1, and E-selectin expressions on HUVECs were significantly lower when morphine was co-treated with LPS than not co-treated. Thus, we suggest that morphine affects the expressions of adhesion molecules primarily by attenuating LPS stimuli on tumor cells.

Treatment of Phalangeal Bone Defect Using Autologous Stromal Vascular Fraction from Lipoaspirated Tissue

PURPOSE: Adipose-derived stromal cells (ASCs) are readily harvested from lipoaspirated tissue or subcutaneous adipose tissue fragments. The stromal vascular fraction (SVF) is a heterogeneous set of cell populations that surround and support adipose tissue, which includes the stromal cells, ASCs, that have the ability to differentiate into cells of several lineages and contains cells from the microvasculature. The mechanisms that drive the ASCs into the osteoblast lineage are still not clear, but the process has been more extensively studied in bone marrow stromal cells. The purpose of this study was to investigate the osteogenic capacity of adipose derived SVF cells and evaluate bone formation following implantation of SVF cells into the bone defect of human phalanx. METHODS: Case 1 a 43-year-old male was wounded while using a press machine. After first operation, segmental bone defects of the left 3rd and 4th middle phalanx occurred. At first we injected the SVF cells combined with demineralized bone matrix (DBM) to defected 4th middle phalangeal bone lesion. We used P (L/DL)LA [Poly (70L-lactide-co-30DL-lactide) Co Polymer P (L/DL)LA] as a scaffold. Next, we implanted the SVF cells combined with DBM to repair left 3rd middle phalangeal bone defect in sequence. Case 2 was a 25-year-old man with crushing hand injury. Three months after the previous surgery, we implanted the SVF cells combined with DBM to restore right 3rd middle phalangeal bone defect by syringe injection. Radiographic images were taken at follow-up hospital visits and evaluated radiographically by means of computerized analysis of digital images. RESULTS: The phalangeal bone defect was treated with autologous SVF cells isolated and applied in a single operative procedure in combination with DBM. The SVF cells were supported in place with mechanical fixation with a resorbable macroporous sheets acting as a soft tissue barrier. The radiographic appearance of the defect revealed a restoration to average bone density and stable position of pharyngeal bone. Densitometric evaluations for digital X-ray revealed improved bone densities in two cases with pharyngeal bone defects, that is, 65.2% for 4th finger of the case 1, 60.5% for 3rd finger of the case 1 and 60.1% for the case 2. CONCLUSION: This study demonstrated that adipose derived stromal vascular fraction cells have osteogenic potential in two clinical case studies. Thus, these reports show that cells from the SVF cells have potential in many areas of clinical cell therapy and regenerative medicine, albeit a lot of work is yet to be done.

Aldosterone Receptor Blockade Prevents Inflammatory Reaction on Type 2 Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: Aldosterone induces renal injury independent of angiotensin II. This harmful effect might be mediated via inflammatory reaction. Aldosterone receptor blockade can retard renal damage in various renal diseases including diabetic nephropathy. However, it is not clear which mechanism is related to the beneficial effect of aldosterone receptor blockade in diabetic nephropathy. Therefore, we investigated whether aldosterone receptor blockade, spironolactone, inhibited inflammatory changes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes. METHODS: To determine the inflammatory effects, urinary MCP-1 protein was measured by ELISA, and intrarenal MCP-1 mRNA and ED-1 expression were examined by RT-PCR and immunohistochemistry, respectively. RESULTS: Blood glucose concentration were higher in diabetic rats than in control rats. Urinary protein excretion was significantly higher in diabetic rats compared with controls since twenty weeks, and proteinuria of the diabetic rats was decreased by spironolactone treatment. Urinary excretion of monocyte chemoattractant peptide-1 (MCP-1) was rapidly increased at the early period in diabetic rats. Spironolactone suppressed urinary level of MCP-1 compared to untreated diabetic rats. Immunohistochemistry revealed a significant increase in ED-1 staining in the diabetic kidney, and spironolactone treatment significantly suppressed intrarenal ED-1 expression in diabetic rats. CONCLUSION: Aldosterone receptor blockade, spironolactone, suppressed proteinuria and inflammatory changes in diabetic rats. These results suggest that spironolactone may have an anti-inflammatory effect in diabetic nephropathy.

The Anti-inflammatory Effect of Retinoid on Streptozotocin-induced Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: An inflammatory mechanism has been suggested to contribute to the progression of diabetic nephropathy. Although retinoid, a known anti-inflammatory agent, has been reported to be beneficial in some experimental renal diseases, it has not been shown whether it prevents disease progression in diabetic nephropathy. Therefore, we investigated whether all-trans retinoic acid inhibits inflammatory changes and improves renal function during the early stages of diabetic nephropathy in streptozotocin-induced diabetic rats. METHODS: We evaluated anti-inflammatory effect of retinoid on streptozotocin-induced diabetic nephropathy. Anti-inflammatory effect was determined by the expression of monocyte chemoattractant peptide-1 (MCP-1). RESULTS: Urinary protein excretion was significantly higher in diabetic rats at four weeks after the induction of diabetes mellitus compared with controls, and proteinuria in the group with retinoic acid treatment was decreased (1.25+/-0.69 vs. 0.78+/-0.72 mg/mg Cr, p=0.056). Urinary excretion of MCP-1 was rapidly increased at two days after induction of diabetes mellitus in diabetic rats, and further increased until four weeks of age compared with control rats. Retinoic acid treatment suppressed to 30% reduction of the urinary level of MCP-1 compared with vehicle treated diabetic rats (119.3+/-74.2 vs. 78.1+/-62.7 pg/mg Cr, p=0.078). Immunohistochemistry revealed a significant increase in staining for MCP-1 protein in the diabetic kidney, and retinoic acid treatment significantly suppressed intrarenal MCP-1 protein synthesis. CONCLUSION: Retinoic acid suppressed proteinuria and inflammatory changes in diabetic rats. These results suggest that retinoic acid may have an anti- inflammatory effect in diabetic nephropathy.

Role of Transforming Growth Factor beta Induced by Podocyte in the Pathogenesis of Diabetic Nephropathy / 대한신장학회잡지

BACKGROUND: According to the development of methods in podocyte cell culture several studies for the role of podocyte in the progression of glomerulosclerosis have been recently reported. But there is few report for the regulation of TGFbeta1 synthesis and type IV collagen production in podocyte in diabetic nephropathy. We investigated the effects of high glucose and TGFbeta1 in culture medium on TGFbeta1 and type IV collagen production and whether their production is dependent on protein kinase C (PKC) pathway in cultured mouse podocyte cell line. METHODS: Conditionally immortalized mouse podocytes with a temperature-sensitive variant of SV40 large T antigen were cultivated. To propagate podocytes, cells were cultivated at 33 degrees C and treated with gamma-interferon (permissive condition). And to induce differentiation, podocytes were changed at 37 degrees C and deprived of gamma-interferon (non-permissive condition). The effects of high glucose and TGFbeta1 in culture media on procollagen alpha1 (PCalpha1 (IV)) and their relationships to PKC pathway were examined. The mRNA expression and protein production of TGFbeta1 and PCalpha1 (IV) were assayed by reverse transcription - polymerase chain reaction (RT-PCR) and western analysis. RESULTS: Compared with normal glucose (NG, 5.5 mM), high glucose exposure (HG, 15, 30 mM) increased the mRNA expression and protein production of TGFbeta1 and PCalpha1 (IV), but without a statistic significance: TGFbeta1 (after 6 hours: 69.62+/-9.00 vs 83.48+/-7.82 vs 74.49+/-24.73, after 24 hours: 65.06+/-20.55 vs 68.01+/-24.35 vs 94.23+/-13.14), PCalpha1 (IV) (after 6 hours: 109.94+/-10.43 vs 102.00+/-6.68 vs 138.65+/-39.83, after 24 hours: 105.88+/-9.53 vs 83.95+/-1.12 vs 109.14+/-3.29, after 72 hours: 99.18+/-5.30 vs 92.93+/-6.33 vs 109.25+/-4.11). TGFbeta1 significantly decreased the expression of PCalpha1 (IV). Calphostin C treatment further stimulated the increase of PCalpha1 (IV) production induced by HG and inhibited the decreased mRNA expression of PCalpha1 (IV) induced by TGFbeta1 administration. CONCLUSION: We suggest that TGFbeta1 have an important role in podocyte in the pathogenesis of diabetic nephropathy. The HG-induced increases of procollagen alpha1 type IV collagen seems to be negatively regulated by TGFbeta1 and PKC pathway and possibly another pathway will positively regulate the production of PCalpha1 (IV), and these pathways may have a different effect on collagen synthesis dependent on the renal cell type.