Jianhua Gao

Improvement of the Survival of Human Autologous Fat Transplantation by Using VEGF-Transfected Adipose-Derived Stem Cells

Background: The efficacy of autologous fat transplantation is reduced by fat absorption and fibrosis due to fat necrosis. Enhanced transplant neovascularization early after transplantation may reduce these outcomes. The authors asked whether cell and concomitant gene therapy using adipose-derived stem cells transduced with vascular endothelial growth factor (VEGF) improves fat transplant neovascularization and survival. Methods: Human adipose-derived stem cells were expanded ex vivo for three passages, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI), and transduced with VEGF or left untransduced. Human fat tissues were then mixed with the DiI-labeled VEGF-transduced adipose-derived stem cells, the DiI-labeled adipose-derived stem cells, the known vascularization-promoting agent insulin, or medium alone, and 18 nude mice were injected subcutaneously with all four preparations, with each of the four designated spots receiving one of these four mixtures in a random fashion. Six months later, transplanted tissue volume and histology were evaluated and neovascularization was quantified by counting the capillaries. Results: Control transplant survival was 27.1 ± 8.2 percent, but mixture with the VEGF-transduced and VEGF-untransduced stem cells significantly increased transplant survival (74.1 ± 12.6 percent and 60.1 ± 17.6 percent, respectively). Insulin was less effective (37.7 ± 6.9 percent). Histological analysis revealed both types of transplants consisted predominantly of adipose tissue, unlike the control transplants, and had significantly less fat necrosis and fibrosis. The VEGF-transduced, adipose-derived stem cell–treated transplants had significantly higher capillary density than the other transplants and bore DiI-double-positive and CD31-double-positive cells (i.e., adipose-derived stem cell–derived endothelial cells). Conclusion: Adipose-derived stem cells together with VEGF transduction can enhance the survival and quality of transplanted fat tissues.

Hypoxia preconditioned human adipose derived mesenchymal stem cells enhance angiogenic potential via secretion of increased VEGF and bFGF.

Mesenchymal stem cells (MSCs) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSCs improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSCs are cultured under ambient or normoxic conditions (21% O2). However, the physiological niches of MSCs have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSCs cultured in standard conditions must adapt from 21% O2 in culture to <1% O2 in ischemic tissue. We have examined the effects of hypoxia preconditioning (1% O2) in human adipose derived mesenchymal stem cells (AD‐MSCs) to discover the conditions that best enhance their tissue regenerative potential. We demonstrate that AD‐MSCs respond positively to hypoxia compared with normoxia preconditioning, show decreased apoptosis even in severe microenvironmental conditions (such as a low‐serum medium), and an increased expression of the angiogenic factors, vascular endothelial growth factor and basic fibroblast growth factor. Human umbilical vein endothelial cells have higher vitality and lower apoptosis when cultured in medium taken from hypoxia‐preconditioned AD‐MSCs, as well as significantly increased capillary‐like structures with this medium on Matrigel. The data suggest that hypoxia preconditioned AD‐MSCs can improve tissue regeneration.

Clinical and basic research on occipito-cervico-dorsal flaps: including a study of the anatomical territories of dorsal trunk vessels.

The authors have carried out a clinical study of all the patients who underwent reconstructions with occipito-cervico-dorsal flaps in their department between 1994 and 2003 and analyzed the outcomes of the surgery. The reconstructed areas ranged from the cheek to the anterior chest. Twenty-eight cases underwent reconstruction with microvascular augmented occipito-cervico-dorsal flaps, and four were reconstructed with single pedicle occipito-cervico-dorsal flaps. In five cases, distal partial necrosis was observed. The largest flap size was 43 × 23 cm (5 × 5-cm pedicle). In the microvascular augmented occipito-cervico-dorsal flaps, the circumflex scapular artery and veins were used in 28 cases, and dorsal intercostal perforators were used together with circumflex scapular artery and veins in five cases. The follow-up term was between 1 and 8 years. Neck scar contractures were released in all cases, and good results were obtained not only functionally but also aesthetically. In an anatomical study, the authors used 20 preserved cadavers and took angiograms of the dorsal region. Five cadavers were used to confirm the territory of each of the vessels that have close relations to the occipito-cervico-dorsal flap (the occipital artery, transverse cervical artery, circumflex scapular artery, and dorsal intercostal perforator artery). Each anatomical territory was clearly seen and its area identified.

Supplementing fat grafts with adipose stromal cells for cosmetic facial contouring.

Background Numerous methods have been proposed to enhance the survival of fat grafts, but no definitive treatment is available. Stromal vascular fraction (SVF)‐assisted cell therapy offers new perspectives for improving fat graft survival. Objectives To determine whether SVF supplementation could improve graft retention in patients undergoing autologous fat grafting for cosmetic improvement of facial contour. Methods We retrospectively analyzed data from 38 women who underwent fat transplantation with SVF (n = 26) or fat grafting alone (n = 12) between October 2010 and January 2012. Each patient underwent computed tomography, and photographs were taken before and 6 months after surgery. The Philips Extended Brilliance Workspace was used for analysis of volume augmentation. Results All patients showed cosmetic improvements, but the degree varied. No complications were evidenced during follow‐up. Fat survival was higher with SVF (64.8 ± 10.2%) than fat grafting alone (46.4 ± 9.3%) (p < .01). SVF supplementation resulted in better clinical improvement than fat grafting alone. Conclusion Supplementing fat grafts with SVF for cosmetic facial contouring can improve the survival of fat grafts over fat grafting alone and provides satisfactory outcomes without major complications. Autologous fat grafting has been used for various cosmetic treatments and difficult reconstructive indications such as temporal depression, wrinkles of nasolabial folds, and hemifacial atrophy, with no incisional scar or complications associated with foreign materials, although problems such as a low rate of graft survival because of early resorption remain. (Aesthet Plast Surg, 14, 1990 and 127) Despite many innovations to overcome these problems, (Dermatol Surg, 26, 2000 and 1159); (Ann Plast Surg, 60, 2008 and 594); (Dermatol Surg, 27, 2001 and 819); (Dermatol Surg, 28, 2002 and 987) we lack a definitive method of fat processing that ensures maximal graft take and viability. (Plast Reconstr Surg, 115, 2005 and 197); (Dermatol Surg, 37, 2011 and 619)

Biological differences between fibroblasts derived from peripheral and central areas of keloid tissues.

Background: Clinical observations indicate that the bulging and reddish peripheral areas of keloids are more elevated than their central areas. Moreover, the peripheral areas of keloids undergo aggressive growth and invasion into normal skin, beyond the boundaries of the initial wound. The aim of this study was to investigate the biological differences between peripheral and central keloid areas. Methods: Six patients suffering from keloids on the anterior chest were selected for this study. Fibroblasts were harvested from both central and peripheral keloid areas. Cell cycle distribution and apoptosis induction were analyzed by flow cytometry and with an antibody to Fas. The expression of apoptosis-related proteins (Fas, Bcl-2, and p53) was measured by flow cytometry. Results: Fibroblasts derived from both central and peripheral parts of keloids displayed significant resistance to Fas-mediated apoptosis. Analysis of cell cycle distribution indicated that approximately 60 percent of fibroblasts derived from the peripheral parts of keloids were in the proliferative periods of the cell cycle (G2 and S phase). However, the majority of fibroblasts derived from keloid centers were in G0 or G1 phase. Fas and Bcl-2 expression did not differ significantly between the two groups, but p53 expression was much higher in fibroblasts derived from central parts than from peripheral parts. Conclusion: It is suggested that differences in cell cycle distribution and p53 protein expression may account for the different growth characteristics of keloid peripheries and centers.

Adipocyte regeneration after free fat transplantation: promotion by stromal vascular fraction cells.

Our objective was to explore the mechanism of cell-assisted adipose transplantation by using freshly isolated human stromal vascular fraction (SVF) cells and to observe the dynamic changes of the graft after transplantation. The SVF was isolated from human liposuction aspirates, and 0.5 ml adipose tissue was mixed with 1 × 106 SVF cells or culture medium then injected to nude mice subcutaneously. At 1, 4, 7, 14, 30, 60, and 90 days after transplantation, samples were harvested for 1) general observation and retention rate; 2) whole-mount stain; 3) H&E stain; 4) immunohistochemical staining for S100, CD68, and CD34; 5) ELISA for VEGF and bFGF; 6) peroxisome proliferator-activated receptor-γ (PPARγ) fluorescence in situ hybridization. The retention rate in the experiment group was markedly higher than that in the control group. Whole-mount stain shows most of the cells in the center of the graft could not survive the ischemia until day 14. Histology showed new vessels on the surface of the graft at 3 days. However, in the control group, fewer newly formed vessels were detected until day 7. In the late stage of transplantation, gradual fibrosis was found in the graft, and the tissue was divided into a grid-like structure. A large number of round neonatal adipocytes with big nuclei in the center were found surrounding the new vessels, which were S100 and CD34 positive and CD68 negative. In the late stage of transplantation, most of the neonatal adipocytes were human PPARγ positive. Moreover, the SVF group showed a higher level of VEGF and bFGF. SVF assisting adipose transplantation could increase the retention rate of the graft through promoting adipose tissue regeneration via secretion of growth factors, promotion of angiogenesis, and increasing the density of mesenchymal stem cells.

Fas-mediated apoptotic signal transduction in keloid and hypertrophic scar.

Background: The pathogenesis of keloid and hypertrophic scar is less well understood because of the lack of animal models. The cell-surface Fas receptor, which is widely distributed in skin components, has been shown to be an important factor that induces apoptosis in human dermal fibroblasts. In this report, the authors performed a comparative study on apoptotic signal transduction in fibroblasts derived from keloids and hypertrophic scars. Methods: Fibroblasts harvested from 10 patients with keloids and hypertrophic scars were used for this study. At first, apoptosis induction using Fas antibody and C2-ceramide were evaluated using electron microscopy and flow cytometry. Second, the expression of the apoptosis-related proteins Fas and Bcl-2 were measured by means of flow cytometry. Third, ceramide was measured by quantitative derivatization to ceramide-1-phosphate using diacylglycerol kinase and [&ggr;-32P] ATP. Moreover, intercellular Ca2+ was investigated using confocal microscopic analyses. Results: In contrast to hypertrophic scar–derived fibroblasts, those derived from keloids are significantly resistant to Fas-mediated apoptosis. The intercellular ceramide and Ca2+ were not activated. There were no significant differences in the level of expression of Bcl-2 between the two groups, but Fas expression was higher in keloid than in hypertrophic scar. Fibroblasts from the two groups were susceptible to ceramide-induced apoptosis. Conclusions: Blocking of the Fas-mediated apoptotic pathway in keloids occurs upstream of the second messenger. The abnormal Fas-induced apoptosis in keloids may account for the imbalance of proliferation and apoptosis. Enhancement of Fas sensitivity could be a promising therapeutic target.

Role of adipose-derived stem cells in enhancing angiogenesis early after aspirated fat transplantation: induction or differentiation?

Autologous fat tissue has been used as a potential filler for soft‐tissue defects, despite unpredictable clinical outcomes and low graft survival. Co‐transplantation of adipose‐derived stem cells (ASCs) is an alternative therapeutic approach to effectively enhance the survival and quality of transplanted fat tissue by increasing neovascularization. Nevertheless, the mechanisms by which ASCs exerted their angiogenic effects remain obscure. ASCs can secrete several angiogenic growth factors, for example vascular endothelial growth factor, hepatocyte growth factor and basic fibroblast growth factor. Hypoxic conditions may promote the proliferation of ASCs and their secretion. However, the differentiation of ASCs into endothelial cells (ECs), pericytes and smooth muscular cells in vivo has not been confirmed. The role of ASCs early after aspirated fat transplantation may be to induce new vessels from the recipient region to grow around and into the graft by releasing significant amounts of angiogenic growth factors rather than to differentiate into ECs, pericytes or smooth muscular cells forming new vessels, an effect that might be enhanced by hypoxia.

Adipose Extracellular Matrix/Stromal Vascular Fraction Gel: A Novel Adipose Tissue-Derived Injectable for Stem Cell Therapy.

Background: Adipose-derived stem cells and other stromal vascular fraction cells were used more often for stem cell therapy, even though limitations such as poor cell retention rate, complicated and expensive isolation processes, and the use of specific laboratory equipment need to be overcome. Methods: Here, the authors developed a novel but simple method for generating an injectable mixture of stromal vascular fraction cells and native adipose extracellular matrix. It is a purely mechanical process in which lipoaspirate is processed into an extracellular matrix/stromal vascular fraction gel. The standard processing procedure was established using quantized tests. The therapeutic potential of the product for wound healing was then tested. Results: Extracellular matrix/stromal vascular fraction gel derived from lipoaspirate and processed using a standard Coleman technique, followed by 1 minute of mechanical processing by passage back and forth between two 10-ml syringes at a flow rate of 10 ml/second, showed the highest adipose-derived stem cell and endothelial cell density. The stromal vascular fraction cells within the product also showed potential for multipotent differentiation similar to that of normal fat samples. In addition, the product showed better therapeutic results than stromal vascular fraction cell suspension when used to treat a nude mouse model of wound healing. Conclusions: Extracellular matrix/stromal vascular fraction gel is an autologous injectable derived from native extracellular matrix and is a functional cellular component generated using a simple mechanical process. As such, it may offer a novel mode of tissue repair suitable for clinical application in stem cell therapies.

Self‐synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber

The development of an engineered adipose tissue substitute capable of supporting reliable, predictable, and complete fat tissue regeneration would be of value in plastic and reconstructive surgery. For adipogenesis, a tissue engineering chamber provides an optimized microenvironment that is both efficacious and reproducible; however, for reasons that remain unclear, tissues regenerated in a tissue engineering chamber consist mostly of connective rather than adipose tissue. Here, we describe a chamber‐based system for improving the yield of mature adipose tissue and discuss the potential mechanism of adipogenesis in tissue‐chamber models. Adipose tissue flaps with independent vascular pedicles placed in chambers were implanted into rabbits. Adipose volume increased significantly during the observation period (week 1, 2, 3, 4, 16). Histomorphometry revealed mature adipose tissue with signs of adipose tissue remolding. The induced engineered constructs showed high‐level expression of adipogenic (peroxisome proliferator‐activated receptor γ), chemotactic (stromal cell‐derived factor 1a), and inflammatory (interleukin 1 and 6) genes. In our system, the extracellular matrix may have served as a scaffold for cell migration and proliferation, allowing mature adipose tissue to be obtained in a chamber microenvironment without the need for an exogenous scaffold. Our results provide new insights into key elements involved in the early development of adipose tissue regeneration.