Frozen Fat Grafts Maintain Vascular Endothelial Growth Factor Expression and Mediate Angiogenesis During Adipose-Derived Stem Cell Enrichment for Soft Tissue Augmentation.

BACKGROUND: Fresh fat grafts are commonly used in both esthetic and reconstructive surgeries, but the graft resorption rate varies. Cryopreservation of unused fat for later touch-up is one option to resolve this variation. In our previous studies, we found that fat cryopreservation may be a practical strategy for storing fat tissue. To explore the cryopreservation method, we evaluated the role of vascular endothelial growth factor (VEGF) in human frozen fat grafts. METHODS: The concentration of VEGF in human frozen fat grafts subjected to different preservation times was determined using Western blotting and enzyme-linked immunosorbent assay. The angiogenic effect of frozen fat grafts was evaluated using a chorioallantoic membrane assay. Furthermore, the impact of adding human adipose-derived stem cells (hADSCs) or different concentrations of avastin (bevacizumab) to frozen fat grafts on angiogenesis was assessed. The viability of frozen fat grafts with or without hADSCs was evaluated using a nude mouse implantation study. Explanted fat tissues were examined on days 1, 4, 7, 14, 28, and 90, and morphological and histological analyses, immunohistochemistry, and enzyme-linked immunosorbent assay (VEGF concentration) were carried out. RESULTS: No significant difference in VEGF concentration between fresh and frozen fat was observed with respect to preservation duration. In the chorioallantoic membrane assay, frozen fat grafts with hADSCs displayed significantly enhanced angiogenesis. Avastin was found to decrease angiogenesis in frozen fat grafts. However, in the nude mouse implantation study, frozen fat grafts displayed VEGF maintenance, with the highest concentration observed on day 7. Adding hADSCs to the graft further increased the VEGF concentration and CD31 expression. Fat graft viability was found to be higher in the frozen fat grafts containing hADSCs than in grafts without hADSCs. CONCLUSIONS: Human fat grafts can maintain VEGF expression under frozen conditions for at least 12 months. The addition of hADSCs to the frozen fat graft could further enhance angiogenesis, VEGF expression, and fat cell viability.

Detection of free flap pedicle thrombosis by infrared surface temperature imaging.

BACKGROUND: Reliable detection of any circulatory issue threatening flap viability after free flap surgery is essential for prompt flap salvage. Currently, the gold standard of flap monitoring is clinical monitoring. However, this method presents logistical challenges to insufficient trained personnel. Auxiliary methods are becoming increasingly vital. MATERIALS AND METHODS: Twelve swine pedicle myocutaneous flaps were harvested and monitored using infrared cameras to investigate the developed monitoring parameters and vascular thrombosis in the free flap model. RESULTS: The mean flap surface temperature after vein or artery occlusion decreased significantly, but the differences were relatively small. As a result, the difference between recorded (flap surface temperature [Ts]) and predicted (estimated surface temperature [Tes]) flap surface temperature (ΔT = Ts- Tes) was used as the parameter for pedicle thrombosis. A ΔT of <0.86°C was used as a vascular occlusion criterion; the sensitivity and specificity of this parameter were 90% and 81%, respectively. The standard deviation of the surface temperature (SDT) was another indicator of vascular occlusion; the estimated sensitivity and specificity for vessel occlusion of SDT < 0.48°C were 84% and 73%, respectively. CONCLUSIONS: Infrared thermal imaging has the advantages of being noninvasive, contact-free, continuous, and able to detect the whole flap surface area. Two indicators, ΔT and SDT, can be used with high sensitivity and specificity for early prediction of flap pedicle thrombosis. Further human studies are necessary to validate clinical application of infrared thermal imaging.

Fabrication of quantum dot-conjugated collagen/hyaluronic acid porous scaffold.

A quantum dot (QD)-conjugated collagen-hyaluronic acid (HA) porous scaffold was combined with our previously reported animal model of mice inferior epigastric flaps and QD infusion to study scaffold angiogenesis. A CdSe/ZnS QD-labeled collagen/HA scaffold was fabricated and examined with a confocal microscope. The degradation rate of the scaffold was inversely related to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide concentration used in cross-linking. There was no cytotoxicity of the QDs as determined by MTT colorimetric assay. Results of the animal implantation study revealed no difference in acute tissue inflammation between scaffolds with or without QD labeling. To study scaffold angiogenesis, we infused the implanted QD-conjugated collagen/HA scaffold with QD of different fluorescence, which can be simultaneously identified by confocal microscope. By these evaluations, we conclude that QD-conjugated collagen/HA porous scaffold is not different from that without conjugation and can be used in our animal model of scaffold angiogenesis without compromising results.

Intra-arterial injection of human adipose-derived stem cells improves viability of the random component of axial skin flaps in nude mice.

BACKGROUND: Skin flap necrosis is a common postoperative complication in reconstructive surgery. Recent evidence suggests that subcutaneously injected adipose-derived stem cells (ASCs) increase the viability of random skin flaps. Here, we examined whether intra-arterial human ASC administration could improve random component survival of axial skin flaps in nude mice. METHODS: Human ASCs isolated from a healthy volunteer by liposuction were injected into nude mice through the right femoral artery at a low (1 × 103 cells), medium (1 × 104 cells), or high (1 × 105 cells) dose. After ASC infusion, right superficial inferior epigastric vessels were ligated to create unipedicled superficial inferior epigastric artery (SIEA) flap with random extension. RESULTS: Flap survival was higher in mice from all three ASC-treated groups, and particularly the medium-dose group was 30% better, than in the control group. Histological examination demonstrated a significantly higher vascular density in the axial skin flap in nude mice treated with the medium ASC dose than in control mice. PKH26-labeled ASCs were identified in skin flaps of ASC-treated mice; some endothelial cells exhibited positive staining for human HLA-A. Compared to the control group, mice in ASC-treated groups had higher vascular endothelial growth factor levels and lower tumor necrosis factor α, interferon γ, and interleukin-6 levels. CONCLUSIONS: Intra-arterial human ASC administration increased the survival of axial skin flaps by attenuating inflammatory reactions and enhancing neovascularization. Intra-arterial ASC administration might yield a higher rate of these cells and of engraftment in the skin flaps. This approach may have a therapeutic role in increasing flap survival.

Adipose-derived stem cells-induced burn wound healing and regeneration of skin appendages in a novel skin island rat model.

BACKGROUND: The study of effectiveness of adipose-derived stem cells (ASCs) in treating burn wounds is still a developing field. The process of wound contraction in areas of loose skin is a major confounding factor in the evaluation and study of burn wound healing in animal models. METHODS: To evaluate the effect of local ASCs administration, deep partial thickness burn wounds were induced by 30 s application of hot copper plates in a novel skin island burn wound rat model to avoid interference from primary wound contraction. Skin islands were divided into two treatment groups-control group (n = 9) injected with PBS and ASCs-treated group (n = 9) injected with 5 × 10 ASCs intradermally. Progress in wound healing was checked at regular intervals after injury (on 1st, 2nd, 3rd, and 4th week) by measuring the mean wound area and analyzing the wound histologically and immunohistochemically, after unstitching the overlaying skin to expose the skin island. RESULTS: It was found that local intradermal injection of ASCs improved burn wound healing at all given time points when compared with control groups, especially in the first 2 weeks (p < 0.05). The percentage of live follicles increased gradually in the ASCs-treated groups compared with control groups between the 3rd and 4th weeks (p < 0.05). The vascular density and proliferating cell nuclear antigen index were also significantly increased in the ASCs-treated groups. CONCLUSION: Thus, in this study, a novel burn wound rat model with reduced interference from wound contraction has been put forth to investigate the therapeutic effects of local administration of ASCs on burn wound healing. Local injection of ASCs not only improved burn wound recovery but also enhanced angiogenesis and skin appendage regeneration.

Decellularized porcine coronary artery with adipose stem cells for vascular tissue engineering.

BACKGROUND: Decellularized xenogenic vascular tissue has potential applications in small-diameter tissue engineering vascular grafts. Decellularization removes most xenogenic antigen and leaves most of the extracellular matrix for cell adhesion, migration and proliferation. Recellularization is recognized as an important step to improve the endothelialization of decellularized vascular grafts in vivo and most studies used endothelial cells for recellularization. However, there have been no studies on applying undifferentiated adipose stem cells (ASCs) in recellularization. MATERIAL AND METHODS: In this study, we evaluated the feasibility of decellularized porcine coronary artery (DPCA) with ASC recellularization as tissue-engineered vascular grafts by in vitro cell biocompatibility and in vivo aorta repair tests. Porcine coronary artery was decellularized with the enzyme-detergent method and characterized by histology and biochemical methods. In vitro biocompatibility was tested by human and rat adipose stem cells (hASCs/rASCs). In vivo, potential for endothelialization of ASC-seeded DPCA scaffolds were evaluated by rat aorta patch repair model. RESULTS: In vitro, hASCs and rASCs could adhere and maintain cell viability on DPCA scaffold. In vivo, rat abdominal aorta repair model revealed that DPCA with rat ASC seeding had a 100% patency rate. Grossly, there was integration between host tissue and graft tissue, and no leakage or rupture was observed. Histologically, DPCA with rat ASC seeding displayed endothelialization on the luminal side. In addition, the layer structure was preserved with collagen deposition. However, intimal hyperplasia was noted. CONCLUSION: This preliminary study indicates that DPCA with undifferentiated ASC seeding exhibited cell biocompatibility in vitro and endothelialization in vivo. DPCA with ASC recellularization has potential for use in the development of small-diameter tissue engineering vascular grafts.

Adipose-derived stem cell conditioned medium attenuates cisplatin-triggered apoptosis in tongue squamous cell carcinoma.

Autologous fat grafting procedures have noted a markedly increased frequency, not only for cosmetic purposes, but also for deformities after head and neck cancer and breast cancer surgery. Carcinogenesis is always a major concern in cell therapy-related issues. However, there is no literature discussing this issue in head and neck squamous cell carcinoma patients. To evaluate the interaction of tongue cancer cells and adipose-derived stem cells, we performed a series of in vitro experiments. Our results demonstrated that cisplatin significantly reduced the viabilities of SCC­25 and CAL­27 cells in a concentration-dependent manner, but it had low cytotoxicity in cisplatin-resistant CAL­27 (CAR) cells. There was no significant difference in terms of viability among the SCC­25, CAL­27, and CAR cells in the adipose-derived stem cell conditioned medium and control groups. There was also no significant difference in terms of cell migration as determined by wound healing assay of SCC­25, CAL­27, and CAR cells between the adipose-derived stem cell conditioned medium treatment and control treatment. Importantly, the adipose-derived stem cell conditioned medium attenuated cisplatin-triggered cell death in the SCC­25 and CAL­27 cells. Moreover, adipose-derived stem cell conditioned medium markedly inhibited cisplatin-induced apoptotic cell death (sub­G1 phase) in the CAL­27 cells. Western blot analyses indicated that cisplatin-induced reductions in pro­caspase­3, pro­caspase­9, phospho-BAD, phospho-IGF-1R, phospho-AKT, and phospho-ERK in CAL­27 cells were reversed by adipose-derived stem cell conditioned medium supplement. Taken together, we provide evidence that adipose-derived stem cell conditioned medium protects CAL­27 cells from cisplatin-induced cell death, possibly through upregulation of the IGF-1R/AKT/ERK signaling pathway.

Auricular Tissue Engineering Using Osteogenic Differentiation of Adipose Stem Cells with Small Intestine Submucosa.

BACKGROUND: Ear reconstruction remains a challenge for plastic surgeons. A tissue-engineering approach could provide another route for obtaining shape maintenance in neoauricular tissue. METHODS: The authors designed a novel tissue-engineering auricular construct by culturing human adipose stem cells, which differentiated into osteocytes but not chondrocytes, in small intestine submucosa scaffolds. The authors evaluated cell growth potential and mechanical properties. An ear-shaped construct was created in vitro and then implanted in the backs of nude mice. The histology, cellularity, neovascularization, mechanical properties, and ear shape maintenance were investigated. RESULTS: In vitro, human adipose stem cells could be successfully seeded in the small intestine submucosa and differentiated toward osteogenesis. The ear-shaped human adipose stem cell/small intestine submucosa construct could maintain its shape in vivo up to 1 year. Alizarin Red S staining confirmed osteogenic differentiation. CD31 stain showed prominent angiogenesis in the human adipose stem cell/small intestine submucosa construct at 6 months and persistence up to 1 year. h-MHC stain revealed the maintenance of cellularity at 6 months and persistence up to 1 year. The mechanical properties were similar to those of native ear cartilage. CONCLUSION: The authors' study found that the combination of human adipose stem cells and small intestine submucosa could provide a more durable ear-shaped construct in vivo. The mechanical properties, shape, and cellularity were maintained in the constructs for up to 12 months. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 µm in diameter), venous wall (VN; >500 µm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 µm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies involving each vessel wall-resident stromal population.

Adipose-derived stem cells exhibit antioxidative and antiapoptotic properties to rescue ischemic acute kidney injury in rats.

BACKGROUND: Acute kidney injury is a major challenge in critical care medicine, with high rates of in-hospital morbidity and mortality. Stem cell therapy has emerged as an evolving technology that could have a substantial impact on acute kidney injury outcomes in the critical care environment. Therefore, the authors investigated the therapeutic effects of adipose-derived stem cells in ischemic acute kidney injury in rats. METHODS: The authors used an ischemia-reperfusion-induced acute kidney injury rat model. The effects of rescuing acute kidney injury were assessed with regard to different adipose-derived stem cell numbers and various routes of administration compared with sham-operated and phosphate-buffered saline-treated groups. RESULTS: Both intrarenal arterial and intravenous administration of adipose-derived stem cells reduced blood urea nitrogen and creatinine levels, and also decreased the tubular injury score 48 hours after ischemia-reperfusion-induced acute kidney injury in a dose-dependent manner, compared with the phosphate-buffered saline-treated group. In the authors' study, it was determined that the optimal cell number was 5 × 10. Furthermore, adipose-derived stem cell transplantation exhibited antioxidative and antiinflammatory properties to reduce apoptosis and promote proliferation of renal tubular cells. CONCLUSIONS: An optimal number of adipose-derived stem cells administered by means of the intrarenal arterial or the intravenous route effectively rescued ischemia-reperfusion-induced acute kidney injury in rats. Antioxidative and antiapoptotic properties of adipose-derived stem cells to reduce tubular cell injury also merit recognition and further study.