Locally Administered Adipose-Derived Stem Cells ... - SAGE Journals

0963-6897/11 $90.00 + .00 DOI: 10.3727/096368910X520065 E-ISSN 1555-3892 www.cognizantcommunication.com

Cell Transplantation, Vol. 20, pp. 205–216, 2011 Printed in the USA. All rights reserved. Copyright  2011 Cognizant Comm. Corp.

Locally Administered Adipose-Derived Stem Cells Accelerate Wound Healing Through Differentiation and Vasculogenesis Chunlei Nie,* Daping Yang,† Jin Xu,‡ Zhenxing Si,† Xiaoming Jin,§ and Jiewu Zhang* *Department of Head and Neck Surgery, The Third Affiliated Hospital of Harbin Medical University, Harbin, China †Department of Plastic Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, China ‡Department of Cell Biology, Harbin Medical University, Harbin, China §Department of Pathology, Harbin Medical University, Harbin, China Despite advances in wound closure techniques and devices, there is still a critical need for new methods of enhancing the healing process to achieve optimal outcomes. Recently, stem cell therapy has emerged as a new approach to accelerate wound healing. Adipose-derived stem cells (ASCs) hold great promise for wound healing, because they are multipotential stem cells capable of differentiation into various cell lineages and secretion of angiogenic growth factors. The aim of this study was to evaluate the benefit of ASCs on wound healing and then investigate the probable mechanisms. ASCs characterized by flow cytometry were successfully isolated and cultured. An excisional wound healing model in rat was used to determine the effects of locally administered ASCs. The gross and histological results showed that ASCs significantly accelerated wound closure in normal and diabetic rat, including increased epithelialization and granulation tissue deposition. Furthermore, we applied GFP-labeled ASCs on wounds to determine whether ASCs could differentiate along multiple lineages of tissue regeneration in the specific microenvironment. Immunofluorescent analysis indicated that GFP-expressing ASCs were costained with pan-cytokeratin and CD31, respectively, indicating spontaneous site-specific differentiation into epithelial and endothelial lineages. These data suggest that ASCs not only contribute to cutaneous regeneration, but also participate in new vessels formation. Moreover, ASCs were found to secret angiogenic cytokines in vitro and in vivo, including VEGF, HGF, and FGF2, which increase neovascularization and enhance wound healing in injured tissues. In conclusion, our results demonstrate that ASC therapy could accelerate wound healing through differentiation and vasculogenesis and might represent a novel therapeutic approach in cutaneous wounds. Key words: Wound healing; Adipose-derived stem cells (ASCs); Differentiation; Secretion; Vascularization

INTRODUCTION

attempted to promote chronic wound healing by increasing systemic or local levels of tissue growth factors, including transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) (1,20,22, 28,31). Modest satisfactory results in wound healing have been shown with various growth factors application. Nevertheless, technical difficulties in delivery and continuous release of growth factors, and enormous cost limit the application of this therapeutic strategy. There is still a critical need for new methods of efficiently enhancing the healing process to achieve optimal outcomes. Recent studies have reported that stem cell therapy may enhance wound healing. Adult stem cells have a prolonged self-renewal capacity with the ability to proliferate and differentiate into various cell types. Fathke

Wound healing is a complex multifactorial process that involves the interaction of inflammation, reepithelialization, angiogenesis, extracellular matrix (ECM) deposition, and remodeling (15,33). The process of wound healing is essential to prevent the invasion of damaged tissue by pathogens and to maintain the integrity of the normal tissue (15). However, healing of wounds is significantly compromised in a number of medical conditions, such as diabetes and chronic renal failure. The mechanisms of compromised wound healing include prolonged inflammation, decreased cellular infiltration and ECM formation, reduced growth factors, and impaired neovascularization (6). In the past decades, numerous strategies have been

Received January 7, 2010; final acceptance June 18, 2010. Online prepub date: August 18, 2010. Address correspondence to Jiewu Zhang, Department of Head and Neck Surgery, The Third Affiliated Hospital of Harbin Medical University, 150 Ha Ping Road, Harbin 150040, China. Tel: 86-451-86298333; E-mail: [email protected] or Daping Yang, Department of Plastic Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, China. E-mail: [email protected]

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et al. demonstrated that bone marrow mesenchymal cells (BMSCs) contributed to the reconstitution of the dermal fibroblast population and accelerate wound healing (7). However, it is well known that the differentiation potential of BMSCs significantly decreases as donor age increases (29). Adipose-derived stem cells (ASCs) represent an attractive alternative of pluripotent cells with characteristics similar to BMSCs (10,11). Moreover, compared with BMSCs, these cells have several advantages including ease of isolation, less donor morbidity, relative abundance, and rapidity of expansion (5,11,37). Based on these outstanding features, ASCs have been applied as a fascinating cell source for cell transplantation therapy in regenerative medicine (9,26,27,32). Previously, we have proposed that ASCs could accelerate cutaneous wound healing (19). Nevertheless, the precise mechanisms that ASCs perform their contribution to wound healing have not been clearly addressed. In the present study, we implanted ASCs into excisional wounds in normal and diabetic rat, respectively, and examined their effects on wound healing compared with vehicle control medium or fibroblasts. We hypothesized that transplantation of ASCs might have the ability to in situ differentiate into various lineages under wound environment, including endothelial and epithelial cell, and produce various angiogenic cytokines that stimulate angiogenesis. To the end, we tested this hypothesis and provided evidences that ASCs could accelerate wound healing through differentiation and vasculogenesis. MATERIALS AND METHODS Isolation and Culture of ASCs Adult male Lewis rats, average weight of 200–250 g, were provided by the Vital River laboratory animal company (Beijing, China) and used for the preparation of implanted ASCs. All animals received humane care in compliance with The Guide for the Care and Use of Laboratory Animals. The following experimental protocol was approved by the ethical committee of Harbin Medical University. Animals were anesthetized using intraperitoneal injection of 10% chloral hydrate (350 mg/ kg). Inguinal rat fat pads were excised and washed extensively with PBS. They were minced finely and digested with 0.075% type I collagenase (Sigma-Aldrich) in a 37°C shaking water bath for 1 h. The cell suspension was centrifuged at 1200 rpm for 10 min and the cell pellet was resuspended in growth media. Then, the cells were plated onto 100-mm2 tissue culture plates and maintained at 37°C in 5% carbon dioxide. Growth media was changed 24 h after the initial plating and every 3 days thereafter. ASCs were harvested at 90% confluence and passaged into 1:3.

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Flow Cytometric Characterization of ASCs Phenotypes of ASCs were analyzed by fluorescenceactivated cell sorting (FACS) (Becton-Dickinson, NJ, USA). Passage 3 ASCs were resuspended in PBS containing 2% fetal bovine serum at 106 cells per milliliter. These cell aliquots were incubated for 30 min on ice with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies: CD11FITC, CD31-PE, CD44-FITC, CD45-PE, CD29-FITC, and CD90-PE. All antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Isotype-matched normal IgGs were used as controls and data analysis was performed with Cell Quest software. In Vitro Multilineage Differentiation of ASCs The culture-expanded ASCs were tested for their ability to differentiate into adipocytes and osteoblasts. Passage 3 ASCs were incubated in corresponding induction medium for 3 weeks. In briefly, adipogenic differentiation was induced with isobutyl-methylxanthine (0.5 mM), dexamethasone (1 mM), insulin (10 mM), and indomethacine (100 mM) in DMEM containing 10% FBS. Then the cells were fixed in 10% formalin and stained with Oil red O solution to show lipid droplets (SigmaAldrich). For osteogenic differentiation, cells were induced in osteogenic induction medium of DMEM containing 10% FBS, dexamethasone (0.1 mM), ascorbate2-phosphate (0.2 mM), and β-glycerophosphate (10 mM). Osteogenic differentiation was confirmed by alkaline phosphatase activity detection (Sigma). Green Fluorescent Protein (GFP) Transfection of ASCs Preparation of cell infection and lentiviral supernatants was performed as described previously (24,36). Briefly, lentiviral vectors were produced by transient cotransfection of HEK293T cells with three plasmids (Invitrogen). The vector-containing supernatants were harvested 48 h after transfection, filtered, and then stored at −80°C. To transduce ASCs with lentivirus constructs, cells were seeded at a density of 2 × 104 cells/cm2 in sixwell plates. Various volumes (50–400 µl) of CMVeGFP lentivirus suspension and 8 µg/ml polybrene (Sigma-Aldrich) were added to EBM-2 (Cambrex) with 5% FBS in a total volume of 1 ml. Cells were allowed to incubate at 37°C for 12 h and replacing it with 2 ml fresh growth medium. At 3 days after transfection, GFPpositive cells were analyzed and sorted using FACS Vantage SE cell sorter. Preparation of Wound Healing Model and ASCs Transplantation Normal Lewis rats (n = 36) and diabetic rats (n = 36) were used in the model of wound healing. For prepara-

ASCs ACCELERATE WOUND HEALING

tion of the diabetic wound model, the male rats were injected with a single dose of streptozotocin (STZ: 65 mg/kg) in 0.1 M citrate buffer (pH 4.5) after overnight fasting to induce diabetes. Animals showing a blood glucose level nearly threefold higher compared with the control value were considered diabetic. At 2 weeks, normal and diabetic rats were anesthetized and prepared according to standard sterile procedure. An 8-mm punch biopsy tool was used to create two circular, full-thickness cutaneous wounds. A donut-shaped silicone splint was centered on the wound to prevent the wound contraction (8). After the preparation of wound healing model, normal rats and diabetic rats were randomly divided into three groups, including the sham group, ASCs group, and fibroblasts group. A total of 1 × 106 cells in 0.8 ml of PBS was injected intradermally around the wound at eight injection sites in the ASC and fibroblast groups. Animals in the sham group received 0.8 ml of PBS as control. Gross Wound Measurements Digital photographs were taken on the day of surgery and every other day thereafter. Time to wound closure was defined as the time at which the wound bed was completely reepithelialized and filled with new tissue. The wound area was measured by tracing the wound margin and calculating pixel area using Sigma Scan Pro Image analysis software. The percentage of wound closure was calculated as follows: (area of original wound − area of actual wound)/area of original wound × 100%. A wound was considered completely closed when the wound area was grossly equal to zero. Histological Analysis of Wounds Rats were sacrificed at 3, 7, 10, 14, and 28 days, at which times the wounds were excised, bisected, and fixed in 10% formalin. Skin samples underwent routine histological processing with hematoxylin and eosin (H&E). Digital analysis software was used to determine the epithelial gap (EG) and the total area of granulation tissue (GT) from these photomicrographs. EG was defined as the distance between the advancing edges of keratinocyte migration. An EG of zero represented a completely reepithelialized wound. Area of GT was calculated by tracing regions of GT and calculating the pixel area. The total area of granulation was defined as the sum of these regions. Three samples were analyzed at each time point and the average was calculated to determine the amount of EG and GT present. Immunofluorescent Analysis Skin biopsies were embedded with OCT and frozen in liquid nitrogen immediately for fluorescent immunostaining. Frozen sections (6 µm) were blocked in 3%

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normal goat serum/0.3% Triton X-100/0.1% BSA (Sigma Aldrich) in PBS for 20 min at room temperature. The sections were then incubated for 12 h at 4°C with the following primary antibodies (mAb): goat anti-GFP (FITC, 1:100, ab6662, Abcam, USA), mouse anti-CD31 (1:100, sc-53526, Santa Cruz), or mouse anti-pan-cytokeratin (1:100, sc-58826, Santa Cruz). Subsequently, the sections were rinsed with PBS and incubated with goat anti-mouse IgG-TR (Texas Red) secondary antibody (1: 200, sc-2781, Santa Cruz) for 1 h at 37°C. Hoechst 33342 dye (C1026, Beyotime Institute of Biotechnology, Shanghai, China) was used to stain nuclei for 2 min. Finally, sections were examined and photographed with a fluorescence microscope. Vessel Density Assessment For microvessel density determination, frozen sections were analyzed using mouse anti-CD31 primary antibody (Santa Cruz). Goat anti-mouse IgG-TR was used as secondary antibody. The procedure was performed as the previously described. To the end, skin sections were digitized to an image and the number of CD31-positive vessels was quantified using Sigma Scan software across five nonconsecutive tissue sections for each wound. Enzyme-Linked Immunosorbent Assay (ELISA) of Growth Factors Growth factor levels for vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and fibroblast growth factor 2 (FGF2) were determined using ELISA in vitro. Basal DMEM devoid of growth factors was added (10 ml) into 100-mm2 culture plates seeded with 1 × 106 ASCs, and the supernatant was collected at 24, 48, and 72 h, respectively. The VEGF level was determined with quantitative ELISA Kit according to the manufacturer’s instructions (RRV00, R&D Systems Inc). Rabbit anti-HGF antibody (ab83760, Abcam) and rabbit anti-FGF2 antibody (sc-7911, Santa Cruz) were used as primary antibodies for ELISA. Goat anti-rabbit IgG-HRP (horseradish peroxidase) (ab6721, Abcam) was used to recognize the primary antibodies. After substrate solution of hydrogen peroxide (R&D Systems) was added into the plate, the optical density of each well was determined by spectrophotometer under the 450 nm wave length and the concentrations of growth factors were calculated. Western Blot Analysis Western blot was used to measure the protein expression of VEGF, HGF, and FGF2 in skin samples in vivo. Skin tissue harvested from wounds was cut into smaller pieces and washed in sterile PBS. The tissue was centrifuged and resuspended into sample application buffer containing a protease inhibitor cocktail (Roche). Sam-

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Figure 1. Characterization of ASCs. (A) Phenotypic characterization of ASCs was analyzed by fluorescence-activated cell sorting. The data indicated that cultured ASCs were positive for the cell surface markers CD29, CD44, and CD90, while they were negative for the CD11, CD31, and CD45. (B) Primary ASCs exhibited a fibroblast-like appearance as observed under a phase contrast microscope. (C) Fluorescent micrograph of GFP labeled ASCs. (D) ASCs were induced into adipogenic lineage. (E) Adipogenic differentiation was confirmed by Oil Red O staining. (F) ASCs were induced into osteogenic lineage. (G) Osteogenic differentiation was revealed by ALP staining. Scale bar: 100 µm.

ples were boiled for 5 min and electrophoresed on 12% polyacrylamide gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Bio-Rad). After blotting, the membranes were incubated with primary antibodies against VEGF (1:1000, ab70612, Abcam), HGF (1:1000, ab83760, Abcam), FGF2 (1:500, sc-7911, Santa Cruz), and β-actin (1:1000, ab8227, Abcam) overnight at 4°C. Subsequently, they were incubated with HRP conjugated goat anti-Rabbit IgG antibody (1:3000, Abcam) for 1 h at room temperature. The immune complex was visualized by photo detection and quantified using scion image software. Statistical Analysis Data are expressed as mean ± SD. Statistical comparisons between the groups were performed using either Student’s t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s test. A value of p < 0.05 was considered statistically significant. RESULTS Characterization of ASCs To characterize the phenotypes of adherent ASCs, flow cytometry was performed. The results showed that

these cultured cells strongly expressed surface antigens such as CD29, CD44, and CD90. In contrast, no expression of the hematopoietic and endothelial lineage markers (CD31 and CD45) was observed. They were also negative for the integrin CD11, an adhesion molecule characteristically found on leukocytes (Fig. 1A). The adherent cultured ASCs exhibited a fibroblast-like morphology (Fig. 1B). Meanwhile, GFP+ ASCs can be easily identified under a fluorescent microscope (Fig. 1C). After cultured in adipogenic induction media for 3 weeks, more than 90% of cells differentiated into adipocytes that could be stained with Oil red O (Fig. 1D, E). In addition, after osteogenic induction, more than 80% of cells differentiated into osteoblasts as demonstrated by alkaline phosphatase staining (Fig. 1F, G). Locally Administered ASCs Accelerate Wound Closure In normal rat model of wound healing, time to complete wound closure was significantly shortened in the ASCs-treated group. The average time for wound closure in the ASCs group was 11 ± 0.77 days whereas the time in the control group was 15 ± 0.63 days (Fig. 2A). In diabetic rat model, wounds treated with ASCs healed


faster than those in control or fibroblasts group. Median time to closure for ASCs-treated wounds was 18 ± 0.41 days compared with 28 ± 0.58 days in the control group (Fig. 2B). An increased rate of wound closure with ASCs treatment was more evident at postoperative day 7 and 10 in normal rat model (Fig. 2C). At day 15, normal wounds in all groups were nearly healed. Compared with the control group, ASC therapy revealed evident acceleration of healing at day 3, 7, 14, and 21 in diabetic rats. There was no difference for time to closure between control and fibroblasts groups (Fig. 2D). The histological observation showed that tissue regeneration was much greater in the ASCs group compared with control or fibroblasts group (Fig. 3A). The experimental data also indicated ASCs treatment enhanced epithelialization at all time points (Fig. 3B). In addition, the amount of granulation tissue in ASCstreated wounds appeared to be more abundant. Poorly formed granulation tissue was significantly delayed in

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control and fibroblast-treated wounds (Fig. 3C). These results indicated that ASCs significantly accelerated epithelialization and granulation tissue deposition, and thus promoted wound healing. Locally Administered ASCs Contribute to Epidermal Structure and Enhance Wound Epithelialization To determine whether locally administered ASCs were incorporated into healing wounds, GFP+ cells were identified using fluorescent microscopy throughout the various substrata of the epidermis. We found a large number of GFP+ cells were present at different location in ASCs-treated wounds, which might indicate multilineage differentiation potential of ASCs in vivo. Because of the GFP+ cells partially found in the newly formed dermis and epidermis, wound sections were further costained for the epithelial marker cytokeratin and GFP to investigate whether ASCs have epithelial differentiation potential. At 7 days postoperative, engrafted GFP+ cells were seen incorporating into dermal structures in ASC-

Figure 2. Effects of ASCs on time course of wound closure. (A) At postoperative day 0, 3, and 10, gross appearance of the wounds with different treatment was photographed in normal rat. (B) At postoperative day 0, 7, and 14, gross appearance of the wounds with different treatment was photographed in a diabetic rat. (C) Wound measurement of percentage closure and time to closure for each group in normal rats. Time to complete wound closure was significantly shortened in the ASC treatment group. An increased rate of wound closure in ASC-treated group was more evident at postoperative day 7 and 10. (D) In diabetic rat model, ASCs significantly accelerated wound closure compared with control or fibroblasts treatment. *p < 0.05 versus control group. FBs: fibroblasts.

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Figure 3. Effects of ASCs on the rate of epithelialization and granulation. (A) Histological observations of wounds in normal rat showed tissue regeneration was much greater in ASCs group than control group. (B) ASCs significantly enhanced the rate of epithelialization of wounds. There was no significant difference in the epithelial regeneration between control and fibroblasts groups. (C) The newly formed granulation tissues in ASC-treated wounds appeared to be thicker and more abundant compared with control wounds. Scale bar: 100 µm. *p < 0.05 versus control group. FBs: fibroblasts.

treated wounds and expressing positive signals for pancytokeratin. No GFP+ cells were found in control or fibroblast groups, indicating specificity of the GFP immunostaining (Fig. 4). These findings indicated that GFP+ ASCs were incorporated into regenerated epidermal structures and enhance wound epithelialization.

Locally Administered ASCs Differentiate Into Vascular Structure To determine whether ASCs were engrafted into vascular structure, the GFP+ cells were further examined for concomitant staining CD31. GFP+ cells were not de-


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tected in control and fibroblast groups. At postoperative day 7, GFP and CD31 double-positive cells were found in newly formed capillaries, indicating spontaneous differentiation of engrafted ASCs into a vascular endothelial phenotype (Fig. 5). At postoperative day 14, increased numbers of ASCs positive for GFP and CD31 appeared in vascular structures that seemed more mature. In addition, immunofluorescent analysis for CD31 demonstrated that ASCs therapy significantly increased neovascularization of wounds. Compared with the control group, blood vessel density was evidently increased in the ASC-treated group at postoperative day 7 (Fig. 6A). At day 14, mean vessel density in the ASC group was 59.4 ± 2.13 vessels, whereas density in control group was 29.8 ± 1.64 vessels (Fig. 6B). Our data suggest that ASCs could participate in the vasculogenesis process of wound healing through differentiate into vascular endothelial cells.

cess, we examined the levels of growth factors in vitro and in vivo. ELISA showed that significantly higher levels of VEGF and HGF were secreted by ASCs after 72h culture than that after 24-h culture (Fig. 7A, B). FGF2 in supernatant was always maintained at a low level (Fig. 7C). Moreover, in vivo expression of VEGF, HGF, and FGF2 protein in wound samples was demonstrated by Western blot. The data revealed that strong VEGF and HGF protein expressions were observed in ASCtreated wounds, while they were lowly expressed in the control and fibroblast-treated wounds (Fig. 7D, E). In addition, a slight but statistically significant enhancement in FGF2 protein expression was detected in ASCtreated wounds (Fig. 7F). There was no significant difference of protein levels between control and fibroblasts group. These results indicate that ASCs secrete angiogenic cytokines, notably HGF and VEGF, and strongly accelerate wound angiogenesis.

ASCs Increase Angiogenic Factor Levels and Enhance Wound Angiogenesis To identify whether a correlation existed between particular growth factor levels and wound healing pro-

DISCUSSION Wound healing is a complex multifactorial process involving the interaction of inflammation, granulation tissue formation, reepithelialization, and angiogenesis

Figure 4. Differentiation of engrafted GFP-positive ASCs into epithelial cells. Frozen sections of wound biopsies in each group were immunostained with an anti-pan-cytokeratin antibody. Fluorescent micrographs showed that ASCs expressing GFP (green) were incorporated into dermal structures in ASC-treated wounds and expressing positive signals for pan-cytokeratin (red), the first epithelial-specific structural protein. Nuclei were stained with Hoechst 33342 (blue). Green staining indicated the absence of GFP+ cells in control and fibroblast groups. The merged images revealed that the implanted GFP-labeled ASCs had differentiated into epithelial cells expressing cytokeratin. Scale bar: 50 µm.

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Figure 5. Differentiation of engrafted GFP+ ASCs into endothelial cells. Frozen sections of wound biopsies in each group were immunostained with an anti-CD31 antibody. Fluorescent micrographs showed that ASCs expressing GFP (green) were incorporated into vascular structures in ASC-treated wounds and expressing positive signals for CD31 (red). Nuclei were stained with Hoechst (blue). Green staining indicated the absence of GFP+ cells in control and fibroblast groups. The merged images revealed that implanted GFP-labeled ASCs had differentiated into endothelial cells expressing CD31. Scale bar: 50 µm.

(15,33). It has been shown that the healing of wounds could be accelerated through delivery of BMSCs, providing strong evidence that MSCs have the extraordinary plasticity and the ability to secrete growth factors that could promote tissue repair (34). Similar to BMSCs, ASCs are other pluripotent cells that possess stem cell characteristics, such as extensive proliferation capacity, and differentiation potential (9,10,37). Moreover, ASCs have several outstanding features described previously (5,11,37). Recently, it has been reported that ASCs could produce physiologically relevant levels of angiogenic and antiapoptotic growth factors (12,13,18,23). These reports make ASCs look more ideal and prompt us to investigate the effects of these cells on wound healing. In this study, we have successfully isolated and cultured ASCs in vitro. Previous studies showed that ASCs could be efficiently cryoperserved with different solutions, while maintaining their viability and differentiation potential (21). This would enlarge the range of application of ASCs in regenerative therapy. In addition, multilineage differentiation capacity of ASCs has also been confirmed and the result was consistent with previous reports (10,11). In in vivo study, we utilized a rat

model of excisional wound healing to determine the effects of ASCs, which prevents wound contraction and allows wounds to heal by epithelialization and granulation tissue formation (8). The gross observation showed that ASCs significantly promoted wound healing process and reduced the time required for complete closure in both normal and diabetic rats. Furthermore, we found accelerated reepithelialization and increased granulation tissue formation in the ASC group. Especially at day 7 and 10, treatment with ASCs had significant promotion effect on epithelial regeneration of wounds. Newly formed granulation tissues were found to be substantially thick and well developed in ASC-treated wounds. These findings suggested that the enhancement role of ASCs on wound healing is associated with increased epithelialization and granulation tissue deposition. In an attempt to investigate the probable mechanisms by which ASCs enhance wound healing, we applied GFP-expressing ASCs on wound healing to determine whether ASCs engrafted in the wound differentiate along multiple lineages of tissue regeneration in the specific microenvironment. It has been shown that under appropriate culture condition in vitro human ASCs have the potential of epithelial differentiation (2). Consistent


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Figure 6. Effects of ASCs on vascularization of wounds. (A) Immunofluorescent staining of CD31 showed blood vessel structures at 1 week postoperatively. Quantification of blood vessel density is expressed as the average number of CD31+ vessels per highpower field. Compared with control group, blood vessel density in wounds was clearly increased in the ASC group. (B) Representative images of CD31 immunofluorescent staining at 2 weeks. Blood vessel density in the ASC-treated group was significantly higher than that in the control group. Scale bar: 50 µm. *p < 0.05 versus control group.

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Figure 7. Effects of ASCs on VEGF, HGF, and FGF2 levels. (A–C) The results of ELISA showed that significantly increased levels of VEGF and HGF secreted by ASCs were observed after 72-h culture. The FGF2 concentration was maintained at a relatively low level. (D–F) The protein expression of VEGF, HGF, and FGF2 in wound was analyzed by Western blot. Protein levels of VEGF and HGF were significantly elevated in ASC-treated wounds compared with control wounds. There was no significant difference of protein expression between the control group and fibroblast groups. A slight but statistically significant enhancement in FGF2 protein expression was detected in the ASC-treated group. *p < 0.05 versus control group.

with this study, our results showed that GFP+ ASCs in vivo appeared to colocalize with stain against pan-cytokeratin, the first epithelial-specific structural proteins, indicating spontaneous differentiation into epithelial cells. Therefore, locally administered ASCs can accelerate wound epithelialization through contributing to dermal keratinocytes. In addition, our data revealed that engrafted GFP+ cells were observed to costain with CD31, an indicator of vascular endothelial phenotype. The observation provided further support that ASCs can in situ differentiate into endothelial lineage and contribute to neovascular structures. This finding was consistent with previous studies demonstrating hASCs could differentiate into endothelial cells and improve postnatal neovascularization (4). In a recent study, DiI-Labeled ASCs were also found enhancing the blood supply of skin flaps through direct differentiation into endothelial cells (14). Although we did not evaluate whether ASCs engrafted in the wound might differentiate into a vascular smooth muscle phenotype, previous research had reported that ASCs possess the potential of myogenic differentiation in vitro (16). In this study, we demonstrated that an important component of the wound healing effect induced

by ASCs was attributable to multiple differentiation in situ, including endothelial and epithelial lineages. However, our data showed a rapid reduction of GFP+ ASCs in wound at postoperative day 21 and thereafter. The underlying mechanisms may be related with progression of wound healing process. These observations further indicated that in situ differentiation of ASCs was the response of multipotent ASCs to the wound microenvironment, which is probably mediated by paracrine growth factors. The paramount importance of blood supply in the healing of wounds has been long appreciated. Recent research has highlighted the unique role of stem cells in revascularization following injury because of their multiple characteristics (3,30). In our study, increased capillary density after ASC injection around the wound bed was obvious with the naked eye, and this was proven by subsequent immunofluorescent analysis. In contrast, treatment with fibroblasts had no significant effect on vessel density. The formation of new blood vessels is necessary to sustain and enhance newly formed granulation tissue. Thus, increased capillaries might have directly accounted for accelerated wound healing. There are at least two mechanisms involved in contribution of


ASCs to the reconstitution of local blood vessels, including direct differentiation into vascular cells and secretion of angiogenic growth factors. GFP-labeled ASCs differentiated into endothelial cells has been discussed above. Furthermore, considering the previous reports supporting direct angiogenic involvement of stem cells, a variety of growth factors might be produced and released by ASCs through paracrine or autocrine approach (13,18,23). As an important finding of this study, we have successfully detected high levels of VEGF and HGF in supernatants of cultured ASCs in vitro. Western blot assay further demonstrated that wounds treated with ASCs strongly expressed protein levels of VEGF and HGF in vivo. It is well known that VEGF is the most effective and specific growth factor that regulate angiogenesis, while HGF is considered to be another important endothelial growth factor with potential angiogenic and mitogenic effects (3,17,18). The application of these growth factors could increase the rate and degree of granulation tissue and capillary formation, and thus accelerate wound healing. Previous studies showed that HGF as well as VEGF could be used in “therapeutic angiogenesis.” Interestingly, unlike the function of BMSCs in angiogenesis, which VEGF plays an important role, the angiogenic activities of ASCs in tissue repair are significantly mediated by HGF but not VEGF (3). It has been suggested that autocrine FGF2 is critical for self-renewal and increases the multipotentiality of ASCs (25,35). Our data showed a slight but significantly increased FGF2 protein expression in ASC-treated wounds compared with the control wounds. In addition, FGF-2 has been found to stimulate the proliferation of fibroblast and capillary endothelial cells, thus promote angiogenesis and wound repair (20). Our main finding is that ASCs enhance neovascularization when injected into the wound bed. The results revealed that angiogenic growth factors secreted by ASCs play a particular significant role in ASC-mediated accelerated wound healing. In conclusion, this study presented that accelerated wound healing could be achieved by local transplantation of autologous ASCs. Here, we showed that ASCs possess the potential of epithelial differentiation, which leads to accelerated reepithelialization in the wound healing process. Furthermore, our data indicated that ASCs have the capacity of differentiation into endothelial cells and secretion of angiogenic growth factors that contribute to increased neovascularization. Taken together, our results demonstrated that locally administered ASCs, which enhance wound healing through differentiation and vasculogenesis, might represent a feasible therapeutic approach in treatment of clinical wounds. ACKNOWLEDGMENT: This research project was supported by Grants from National Natural Science Foundation of China (30325042).

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