Ziqing Dong

Anti-aging effect of adipose-derived stem cells in a mouse model of skin aging induced by D-galactose.

Introduction Glycation products accumulate during aging of slowly renewing tissue, including skin, and are suggested as an important mechanism underlying the skin aging process. Adipose-derived cells are widely used in the clinic to treat ischemic diseases and enhance wound healing. Interestingly, adipose-derived stem cells (ASCs) are also effective in anti-aging therapy, although the mechanism underlying their effects remains unknown. The purpose of the present study was to examine the anti-aging effect of ASCs in a D-galactose-induced aging animal model and to clarify the underlying mechanism. Materials and Methods Six-week-old nude mice were subcutaneously injected with D-gal daily for 8 weeks. Two weeks after completion of treatment, mice were randomized to receive subcutaneous injections of 106 green fluorescent protein (GFP)-expressing ASCs, aminoguanidine (AG) or phosphate-buffered saline (PBS). Control mice received no treatment. We examined tissue histology and determined the activity of senescence-associated molecular markers such as superoxide dismutase (SOD) and malondialdehyde (MDA). Results Transplanted ASCs were detectable for 14 days and their GFP signal disappeared at day 28 after injection. ASCs inhibited advanced glycation end product (AGE) levels in our animal model as well as increased the SOD level and decreased the MDA level, all of which act to reverse the aging phenotype in a similar way to AG, an inhibitor of AGE formation. Furthermore, ASCs released angiogenic factors in vivo such as vascular endothelial growth factor, suggesting a skin trophic effect. Conclusions These results demonstrate that ASCs may contribute to the regeneration of skin during aging. In addition, the data shows that ASCs provide a functional benefit by glycation suppression, antioxidation, and trophic effects in a mouse model of aging.

Quantitative Volumetric Analysis of Progressive Hemifacial Atrophy Corrected Using Stromal Vascular Fraction–Supplemented Autologous Fat Grafts

BACKGROUND Progressive hemifacial atrophy (Parry‐Romberg disease) is rare and involves all skin layers and subcutaneous soft and hard tissue. Autologous fat grafting has revolutionized the field of soft‐tissue reconstruction and augmentation, but long‐term maintenance is unpredictable. Stromal vascular fraction (SVF)‐supplemented cell therapy offers new hope for improving fat graft survival, with good long‐term results, but efficacy and long‐term outcome in the clinic are rarely studied using objective data. OBJECTIVE To compare the long‐term viability of SVF‐supplemented fat grafts and fat grafts alone for contour reconstruction of progressive hemifacial atrophy using quantitative volume analysis. METHODS We treated 20 patients with stable hemifacial atrophy for at least 2 years with SVF‐supplemented autologous fat grafting (n = 10) or fat grafting alone (n = 10). All patients were followed up every 3 months. Hemifacial volume was measured using computed tomography and the Philips Extended Brilliance Workspace. RESULTS All patients had successful outcomes without complications, but fat survival and clinical improvement was greater with SVF‐supplemented grafting than fat grafting alone after 6 months. CONCLUSION SVF‐supplemented autologous fat transplantation is effective and safe for treating progressive hemifacial atrophy and can enhance the survival of grafts in the face without major complications.

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.

The angiogenic and adipogenic modes of adipose tissue after free fat grafting.

Background: The major drawback of adipose grafting is its clinical unpredictability, which leads to surgeon and patient dissatisfaction. The mechanisms underlying angiogenesis and regeneration of the graft tissue are still unclear. Methods: Mouse adipose tissue was processed using two different methods (fragmental and integral) and was used to identify the mode of angiogenesis of the graft. Cross-grafting of tissue from normal mice and transgenic mice expressing green fluorescent protein was used to observe the origin of cells during the adipose regeneration. Results: Almost all the CD31 endothelial cells of the new vessels were derived from the recipient. The new vessels in the graft were mainly formed through recipient vessels growing into the graft rather than the reassembly of donor endothelial cells or the reconnection of recipient and donor vessels. Angiogenesis depends largely on recipient-site environment. The retention of donor-derived tissue dropped to only 10 percent 8 weeks after grafting, and the majority of the key regeneration cells, the CD34+ cells, came from the recipient during adipogenesis (p < 0.05). In total, the retention of the recipient-derived tissue was up to 73 percent in the fragmental group and 47.5 percent in the integral group. Conclusions: The angiogenesis of the graft occurs by the classic “vessel branching” mode, in which the recipient plays a dominant role. The mode of graft tissue retention primarily involves CD34+ adipose precursor cells derived from the recipient.

Adipose stem cell-laden injectable thermosensitive hydrogel reconstructing depressed defects in rats: filler and scaffold

Facial depressed defects are a common cosmetic problem. Temporary fillers need to be re-injected frequently to maintain the desired outcomes. Here, the feasibility of a novel type of injectable hydrogel for persistent effect is demonstrated. We first useed agmatine to synthesize a poly(amidoamine) (PAA) to form a cell-attachable crosslinker and then the crosslinker was co-polymerized with N-isopropylacrylamide to obtain an injectable and temperature sensitive hydrogel. 1H NMR showed the successful synthesis of the crosslinker. In vitro tests, CCK-8 assay and live/dead viability test showed that the hydrogel was non-toxic to adipose-derived stem cells (ASCs). SEM images also confirmed that ASCs could adhere to the hydrogel. Then we constructed a novel depressed defect model in rats and injected four different fillers in the depressed defects: (1) the hydrogel with ASCs, (2) the hydrogel only, (3) hyaluronic acid, and (4) PBS. After 4 weeks, gross and histological analyses showed the defects in hydrogel, hydrogel + ASCs, and HA groups improved significantly and there were no significant differences among them. Significant differences in thickness from skin to muscle in the defect was found between the hydrogel + ASCs group and the other groups after 6 months. The hydrogels degraded completely in defects in both the hydrogel group and the hydrogel + ASCs group, and were filled with adipocytes and multilocular immature adipocytes. Immunohistochemical study using s-100 and perilipin staining revealed adipocyte differentiation in the defect sites. We also used green fluorescent protein (GFP)-ASCs for tracing and found that exogenous added ASCs were involved in adipogenesis. In conclusion, such a cell attachable thermosensitive hydrogel has definite potential not only as a filler but also as a scaffold, and has a persistent effect for small depressed defects. It might ultimately become a new material in plastic and reconstructive surgery.

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.

The Fate of Fat Grafts in Different Recipient Areas: Subcutaneous Plane, Fat Pad, and Muscle.

BACKGROUND Although fat is transplanted into several layers, including subcutaneous, fat, and muscle layers, there is little (clinical) scientific basis for these procedures. OBJECTIVE To determine the optimal recipient layer for fat transplantation. MATERIALS AND METHODS Fat harvested from inguinal fat pads of green fluorescent protein (GFP) mice was grafted into the subcutaneous and intramuscular planes and the fat pads of C57 mice. Specimens collected after 1, 4, 8, 12, and 16 weeks were stained with hematoxylin and eosin to determine angiogenesis and fibrosis in the grafts. The survival rate of donor adipose tissue was determined by measuring GFP expression. RESULTS Fat was retained longer in fat pads than in subcutaneous layers of recipient mice and longer in subcutaneous than in intramuscular layers. Angiogenesis and vascularized connective tissue were greater in intramuscular than in subcutaneous or fat grafts. Neovascularization, however, was similar in fat pads and subcutaneous grafts. Survival rate was higher for intramuscularly injected fat than subcutaneously and fat pad injected fat. CONCLUSION Fat pad injection showed the highest graft retention rate, indicating that fat pads maybe the optimal area for fat transplantation. The increased blood supply to muscle suggests that intramuscular injection maybe optimal when there is little movement.

The Combination of Tissue Dissection and External Volume Expansion Generates Large Volumes of Adipose Tissue

Background: Noninvasive external volume expansion device has been applied to stimulate nonsurgical breast enlargement in clinical settings. Although previous results demonstrate the capacity of external volume expansion to increase the number of adipocytes, this strategy alone is insufficient to reconstruct soft-tissue defects or increase breast mass. The authors combined a minimally invasive tissue dissection method with external volume expansion to generate large volumes of adipose tissue. Method: In vitro, various densities of adipose-derived stem cells were prepared to evaluate relations between cell contacts and cell proliferation. In vivo, dorsal adipose tissue of rabbits was thoroughly dissected and the external volume expansion device was applied to maintain the released state. External volume expansion without tissue dissection served as the control. Results: In the dissection group, the generated adipose tissue volume was much larger than that in the control group at all time points. A larger number of proliferating cells appeared in the dissection samples than in the control samples at the early stage after tissue dissection. At low cell density, adipose-derived stem cells displayed an increasing proliferation rate compared to high cell density. Protein expression analysis revealed that cell proliferation was mediated by a similar mechanism both in vivo and in vitro, involving the release of cell contact inhibition and Hippo/Yes-associated protein pathway activation. Conclusions: Adipose tissue dissection releases cell-to-cell contacts and induces adipose-derived stem cell proliferation. Preexpanded adipose-derived stem cells undergo adipogenesis under the adipogenic environment created by external volume expansion, leading to better adipose regeneration compared with the control.

In Vivo Dedifferentiation of Adult Adipose Cells

Introduction Adipocytes can dedifferentiate into fibroblast-like cells in vitro and thereby acquire proliferation and multipotent capacities to participate in the repair of various organs and tissues. Whether dedifferentiation occurs under physiological or pathological conditions in vivo is unknown. Methods A tissue expander was placed under the inguinal fat pads of rats and gradually expanded by injection of water. Samples were collected at various time points, and morphological, histological, cytological, ultrastructural, and gene expression analyses were conducted. In a separate experiment, purified green fluorescent protein+ adipocytes were transplanted into C57 mice and collected at various time points. The transplanted adipocytes were assessed by bioluminescence imaging and whole-mount staining. Results The expanded fat pad was obviously thinner than the untreated fat pad on the opposite side. It was also tougher in texture and with more blood vessels attached. Hematoxylin and eosin staining and transmission electron microscopy indicated there were fewer monolocular adipocytes in the expanded fat pad and the morphology of these cells was altered, most notably their lipid content was discarded. Immunohistochemistry showed that the expanded fat pad contained an increased number of proliferative cells, which may have been derived from adipocytes. Following removal of the tissue expander, many small adipocytes were observed. Bioluminescence imaging suggested that some adipocytes survived when transplanted into an ischemic-hypoxic environment. Whole-mount staining revealed that surviving adipocytes underwent a process similar to adipocyte dedifferentiation in vitro. Monolocular adipocytes became multilocular adipocytes and then fibroblast-like cells. Conclusions Mature adipocytes may be able to dedifferentiate in vivo, and this may be an adipose tissue self-repair mechanism. The capacity of adipocytes to dedifferentiate into stem cell-like cells may also have a more general role in the regeneration of many tissues, notably in fat grafting.