Abstract
Cell-based therapy is an attractive approach for the treatment of chronic nonhealing wounds. This study investigated whether adipose-derived stem cells (ASCs) can accelerate diabetic wound healing and traffic in the engraftment of ASCs. Dorsal full-thickness skin wound defects (6 × 5 cm) were created in a streptozotocin (STZ)-induced diabetes rodent model. Group I served as a nondiabetic normal control, group II served as a diabetic control without ASCs, and group III included rats that were injected subcutaneously in the wound margin twice with nondiabetic ASCs (1 × 107 ASCs/dose). The wound healing was assessed clinically. Histological examination and immunohistochemical analyses of periwound tissue were performed. Green fluorescence protein (GFP)+-ASCs were used to examine the engraftment of these cells after injection. XenoLight DiR-labeled ASCs were implanted to detect migration ability using an IVIS imaging system. Results revealed that complete wound healing time statistically decreased in the ASC-treated group compared to the controls (p < 0.001). Histological examination revealed the ASC-treated group showed a significant reduction in the proinflammatory reaction, with significantly increased levels of EGF, VEGF, rPH, and Ki-67 expression compared to the controls. The populations of GFP+-ASCs in circulating blood significantly increased after ASC injection compared to those of controls. Immunofluorescence staining showed GFP+-ASCs significantly accumulated in the subdermal layer of the wound margin and increased angiogenesis via vWF and VEGF expression after injection. IVIS analysis revealed ASCs could exist and home into the periwound area up to 8 weeks postimplantation. In conclusion, ASCs significantly enhanced diabetic wound healing, engrafted into the local wound tissue, and implanted into circulating blood. ASC treatment stimulated neoangiogenesis and increased tissue regeneration through paracrine and autocrine mechanisms.
Introduction
Diabetes mellitus is responsible for delayed or impaired wound healing, which often leads to chronic ulcer formation. Chronic wounds represent a relevant clinical and socioeconomic burden. Diabetes is a chronic disease that involves approximately 350 million people (6.5%) worldwide. The annual incidence of foot ulcers among people with diabetes is estimated at between 1% and 4.1%, and the annual incidence of amputation is 0.21-1.37% (6,13). The amputation rate is 15-70 times higher in diabetic individuals than in the general population (3,19). Many adjunctive treatment options, however, both concomitant and sequential therapeutic approaches, are highly resistant to treatment in some cases and are often ineffective or slowly progressive (6,9,10,14,16,29). As technology advances, several topical treatment strategies have been applied in experimental nonhealing wound models (15,17,26,28). However, although new therapeutic methods have been developed, many chronic wound treatments remain unsatisfactory, and treatment strategies that are more effective must be established.
Optimum cutaneous wound healing requires a well-orchestrated integration of complex biological and molecular events for cell migration, proliferation, extracellular matrix (ECM) deposition, angiogenesis, and remodeling. However, this orderly sequence of cellular and molecular events is disrupted in diabetes and leads to compromised wound healing (11,36). Many factors contribute to non-healing wounds, including impaired cytokine production by local inflammatory cells and fibroblasts. Reduced tissue regeneration and angiogenesis are crucial for wound healing (5,31).
Cell-based therapy is an attractive approach for the treatment of difficult nonhealing wounds (38). This field of regenerative medicine focuses primarily on stem cells, which are specialized cells with the ability to self-renew and differentiate into multiple cell types (8). Adult multipotent stem cells are crucial for physiological tissue renewal and regeneration after injury. Mesenchymal stem cells (MSCs) have been isolated from various sites, including bone marrow, adipose tissue, and amniotic fluid (1,23,32). Previous studies have shown that different types of stem cells, such as bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived stem cells (ASCs), are capable of enhancing wound healing in experimental diabetic models (20,22,30,37). BM-MSCs have demonstrated several properties in vitro that can promote tissue repair, including the production of multiple growth factors, cytokines, collagens, and matrix metalloproteinases (12,32) and the ability to promote the migration of other skin cells, such as keratinocytes (2). Our previous study demonstrated that the topical subcutaneous injection of an optimal dosage of BM-MSCs into the diabetic wound margin enhances the wound healing process in diabetes (25). Our findings suggested that BM-MSCs enhance wound epithelialization and neovascularization via the increased expression of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) and the induction of epithelization in the transitional zone of the wound (25). Although BM-MSCs have demonstrated several properties that promote wound healing, the preparations of BM-MSCs are time consuming.
ASCs are abundant in fatty tissue and have a multilineage mesenchymal potential that is similar to BM-MSCs. Previous studies have indicated that harvesting and culturing MSCs from adipose tissue are easier than collecting cells from bone marrow (30,39). ASCs represent an alternative source of multipotent stem cells with characteristics that are similar to BM-MSCs (4). ASCs are easier to isolate and are relatively abundant, which make these cells a potential source for wound repair and regeneration (20,22,30).
Therefore, in the present study, we investigated whether nondiabetic ASCs could enhance diabetic wound healing and tissue regeneration in a rodent dorsal full-thickness wounding defect model. Additionally, the paracrine and autocrine effects of ASCs that increase cellular proliferation and epithelialization were examined.
Materials and Methods
Animals
All of the animals were treated humanely according to the guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. All of the animals were housed under standard conditions. The Division of Laboratory Animal Resources at Kaohsiung Chang Gung Memorial Hospital (CGMH), Taiwan, administered veterinary care to the rodents. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at CGMH.
Streptozotocin (STZ)-Induced Diabetes
Diabetes was induced in 4-month-old male Wistar rats (BioLASCO Taiwan Co. Ltd., Taipei, Taiwan) by a single intraperitoneal injection of STZ (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) (25,26). All animals were confirmed diabetic (plasma glucose levels of >300 mg/dl) at 1 week after STZ injection. The animals were monitored for 4 weeks for manifestations of the physiological abnormalities of diabetes preoperatively. The diabetic rats were administered intermittent-acting insulin (1-2 unit/kg; Montards Novo Nordisk A/S) subcutaneously to equalize blood glucose levels at 200-300 mg/dl.
Animal Model
The animal model was followed as described in our previous report (26). The dorsum of each rat was shaved, and a 6 × 5-cm patch was drawn on the dorsal skin. The palpable hip joints were used as anatomical landmarks for defining the base of the wound defect. The skin flap was excised to create a full-thickness skin defect with an area of 6 × 5 cm. The margin of the wound defect was sutured in place with 4-0 silk sutures to prevent wound contracture (25,26). Then the wound was temporarily covered with transparent Tegaderm™ (3M HealthCare, Borken, Germany) until ASC therapy was initiated. Following surgery, the rats were returned to their individual cages in the animal holding room after the rats had regained consciousness. The rats were housed separately post-operatively. Antibiotics (ampicillin 50 mg/kg/day; Y F Chemical Corp., New Taipei, Taiwan) were administered routinely to these animals for 3 days postoperation.
ASC Culture
ASC isolation was performed according to our and other published methodologies (24). Groin adipose tissue was minced, washed, and digested with 0.2% collagenase type IA (4 mg/ml; Sigma-Aldrich) for 30 min at 37°C. Next, the suspension was centrifuged at 500 × g for 5 min at room temperature to pellet cells from the stromal vascular fraction. Following RBC lysis with ammonium chloride lysis buffer (155 mM ammonium chloride, 0.1 mM EDTA, and 10 mM sodium bicarbonate, pH 7.2; all from Sigma-Aldrich), the cells were washed with phosphate-buffered saline (PBS, 10 mM sodium phosphate dibasic, 2 mM potassium dihydrogen phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride; all from Sigma-Aldrich), resuspended in maintenance medium, and then plated in culture dishes. ASCs were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) that was supplemented with 10% fetal bovine serum (FBS; Hyclone, South Logan, UT, USA), and 1% penicillin-streptomycin (Invitrogen) and subcultured before confluency. The ASCs were expanded in culture and characterized by flow cytometry following positive surface staining for CD44 (1 mg/ml; BD Pharmingen, San Diego, CA, USA), CD90 (1 mg/ml; BD Pharmingen), MHC class I (1 mg/ml; Affinity Bioreagents, Inc., Golden, CO, USA), and CD106 (1 mg/ml; BD Pharmingen) but not for CD45, MHC class II, and CD80/B7-1. Before experimental use, the ASCs were tested as described previously for their ability to differentiate into various mesenchymal lineages, including adipocytes, osteoblasts, and chondrocytes (25).
Experimental Design
Thirty-six male Wistar rats were divided into three groups. All of the rats were administered 5% isoflurane (Forane, AbbVie, Austria) anesthesia through a calibrated vaporizer intramuscularly. Additionally, the rats received atropine (0.1 mg/kg; Oriental Chemical Works Inc., New Taipei, Taiwan) injections to reduce saliva secretion during and after the surgery. In group I (normal group without diabetes, NC), the dorsal skin defect was created but was not treated with ASCs. In group II (diabetic control group, C), the diabetic dorsal skin defect was created but was not treated with ASCs. In group III (ASC-2), the diabetic dorsal skin defect was created and subsequently injected with syngeneic nondiabetic ASCs twice, once on day 3 and once on day 7 postwounding. The ASCs were applied to eight areas (subcutaneous topical injection of 1 × 107 cells/area for eight areas in all wound edges) along the margin of the dorsal wound. The ASC dose and the optimal timing of the two sessions of ASC injection were modified from our previous study because the study revealed that the wound healing time was faster in two sessions of BM-MSC treatment than in one session of BM-MSC treatment in a rodent wound model (25).
GFP+-ASCs were propagated from the adipose tissue of GFP-transgenic Wistar rats [W-Tg (CAG-GFP) 184Ys], which were kindly provided by the Eiji Koayashi Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan, for visualizing the migration of the implanted GFP-labeled ASC (GFP+-ASC) engraftment in the periwound edge. The isolation and subculture of GFP+-ASCs was performed according to described ASCs culture methodologies (24).
Wound Healing Area Estimation
The wound healing area was assessed once per 3 days postoperation using the described template technique (26). The healed area, which is given by the expression [1 - (A1/A0) x 100%], was calculated from the original wound area (6 × 5 cm2 as A0) and the unhealed area (A1) once per 3 days until the entire wound had healed.
Histological Examination
Full-thickness 3-mm biopsies were performed from the wound margin on days 3 and 7 after the final topical ASC subdermal injection. Biopsy specimens were fixed in 10% formalin (Sigma-Aldrich) and embedded in paraffin. Sections (4 μm thick) for each group were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) for leukocyte infiltration from the dermis to the subcutaneous layers in response to leukocyte-mediated inflammation.
Immunohistochemical Analysis
Semiquantification of immunohistochemical staining was performed using the number of cells with positive horseradish peroxidase-diaminobenzidine (HRP-DAB) staining (26,27) using the Mouse/Rabbit PolyDetector Plus HRP/DAB System (BIO SB, Santa Barbara, CA, USA). Cellular proliferation was assessed by examining the expression of Ki-67 (1:300 dilution; Upstate, New York, NY, USA), leukocyte infiltration was evaluated by detecting the expression of CD45 (1:300 dilution; BD Pharmingen), and tissue remodeling and collagen processing were examined by the expression of prolyl 4-hydroxylase (rPH), which is an enzyme that is involved in collagen biosynthesis (1:300 dilution; Chemicon/Millipore, Temecula, CA, USA). EGF (1:400 dilution) and VEGF (1:500 dilution; Santa Cruz, Biotechnology, Inc., Dallas, TX, USA) expression was examined in the superficial epidermis and dermis and in the underlying subcutaneous layer. Polyclonal antibodies against CD45, Ki-67, rPH, VEGF, and EGF were used as the primary antibodies. Then the sections were incubated with biotinylated goat anti-rabbit antibodies for 60 min. The specific binding of the secondary antibodies (R&D Systems, Minneapolis, MN, USA) to the primary antibodies was visualized using HRP for the enzymatic conversion of the chromogenic substrate 3,3′-diaminobenzidine (DAB) into a brown precipitate using the Cell and Tissue Staining kit (R&D Systems).
Immunofluorescence Staining
Frozen sections were obtained from the periwound edge at day 10 and at 28 weeks after GFP+-ASC implantation. The samples were embedded in Optimal Cutting Temperature Tissue (OTC)-Tek® (Sakura, Fine Technical Co., Tokyo, Japan) and stored at −80°C before use. Ten-micrometer-thick cryosections were adhered to poly-L-lysine slide (Muto Pure Chemicals, Tokyo, Japan) and fixed in acetone (Sigma-Aldrich) for 10 min. Neoangiogenesis was identified by colocalized green and red fluorescence using double-immunofluorescence staining of GFP expression and rhodamine-labeled von Willebrand factor (vWF, 1:200 dilution; Serotec, Oxford, UK) or Texas red-labeled VEGF (1:200 dilution; Jackson Immuno Research Laboratories, West. Grove, PA, USA). The sections were also stained with H&E.
Flow Cytometric Assessment
A flow cytometry-based assay was performed after ASC infusion for 4 weeks to determine whether circulating GFP+-ASCs populations existed in the recipient peripheral blood. The blood cells were incubated with 5 μl of polyclonal antibody against GFP (1 mg/ml; Abcam, Cambridge, UK) to identify infused ASCs. After being incubated for 30 min at room temperature, the cells were centrifuged, washed, and analyzed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were subsequently analyzed using BD FACScan™ software.
In Vivo Fluorescence Imaging for the Detection of XenoLight DiR-Labeled ASCs
XenoLight DiR [DiIC18 (7) or 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide1-labeled ASCs were implanted in six rats on day 3 postwounding and monitored using an IVIS Imaging System 200 Series (Caliper Life Science, Hopkinton, MA, USA) to investigate ASC engraftment and in vivo distribution in the wound area. XenoLight DiR is a lipophilic, near-infrared fluorescent cyanine dye that is ideal for staining the cytoplasmic membrane, enabling the detection of fluorescent stained-ASC cells using an IVIS imaging system. Biofluorescence was analyzed using Living Image 3.10 software (Caliper Life Sciences).
Examination of Histomorphometrical Markers
Tissue sections were imaged using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Gottingen, Germany) to quantify the immunohistochemically stained cells. Four random images from each selected area were acquired at 400x magnification. The number of immunopositive cells and the percentage of positively labeled cells to total cells were presented.
Statistical Analysis
The experimental results are presented as the means ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed to determine the significant differences in groups with a normal distribution. Post hoc comparison was performed using Tukey's HSD tests, and p < 0.05 was considered statistically significant.
Results
ASC Treatment Enhanced Diabetic Wound Healing
The overall results indicated that the wound size was significantly reduced in the ASC-treated rats compared to that of the control (Fig. 1A). The complete wound healing time was statistically shorter in ASC-treated rats compared to that in diabetic controls without treatment (6.8 ± 0.75 weeks vs. 9.8 ± 0.75 weeks, p < 0.001) (Fig. 1B). This result indicated that ASC administration via subdermal injection could accelerate diabetic wound healing.
ASC injection into the subdermal periwound edge decreased the wound healing time in diabetic rats. (A) The wound size was significantly reduced in the ASC-treated groups compared to the controls. The differences in the indicated signals were statistically significant (p < 0.001) between control and ASCs. (B) The complete wound healing time was statistically shorter in ASC-treated rats compared to that in controls (6.8 ± 0.75 weeks vs. 9.8 ± 0.75 weeks, p < 0.001). Abbreviations: NC, normal control; C, diabetes control without treatment; ASC-2, two sessions of ASCs.
ASC Treatment Suppressed the Periwound Inflammatory Responses
H&E staining of the biopsy specimens from the wound edge revealed that leukocyte infiltration from the dermis to the subcutaneous layers exhibited a marked decrease in the leukocyte-mediated inflammatory response following ASC administration on days 3 and 7 compared to that of the controls (Fig. 2). IHC staining analysis indicated the CD45 expression significantly decreased in the ASC-treated group compared with that in the control group (Table 1).
ASCs suppressed the early inflammatory responses in periwound edges. Histological examination indicated that the leukocyte infiltration from the dermis to the subcutaneous layers clearly decreased in the ASC-treated group on days 3 and 10 after ASC treatment compared to that of the controls. Magnification: 100x.
Analysis of Immunohistochemical Positive Labeled Cells in the Wounding Margin
Abbreviations used: CD45, leukocyte common antigen; Ki-67, proliferating cell nuclear antigen; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; rPH, prolyl 4-hydroxylase.
ASC Treatment Promoted Cellular Proliferation and Growth Factor Expression
Cellular proliferation, which was analyzed in terms of the Ki-67 expression levels at the wound edge, revealed a significant increase in Ki-67 expression in the ASC-treated group compared to that of the controls (p < 0.001). In addition, the expression of rat rPH in fibroblasts significantly increased along the wound edge in the ASC-treated group compared to that in the controls (p < 0.001). EGF expression was clearly upregulated in the wound margin, particularly in the fibroblasts and epithelial cells, in the ASC-treated group compared to that in the control group (p < 0.001). The expression level of VEGF in the ASC-treated group significantly increased, particularly in the fibroblasts and endothelial cells, compared to that in the controls (p < 0.001). Semiquantitative IHC staining IH indicated cells positively labeled with CD45, Ki-67, VEGF, EGF, and rPH in the wound margin at 3 days and 10 days posttreatment; the results are summarized in Table 1 (shown as percentages).
ASCs in the Engraftment Migrated
The state of ASCs at the transplanted sites was confirmed by examining the expression of GFP in GFP+-ASCs. Immunofluorescence staining indicated that many GFP+-ASCs accumulated in the subdermal layer of the wound margin at 10 days after periwound subdermal injection. The GFP+-ASCs strongly migrated into the sub-cutaneous layers at 28 days after the local administration of ASCs (Fig. 3). Additionally, flow cytometric analysis revealed that circulating GFP+-ASC populations existed in the peripheral blood after 4 weeks posttreatment with GFP+-ASCs (Fig. 4).
Migrating ASCs were analyzed by examining the expression of GFP-labeled ASCs. Immunofluorescence stain revealed that many GFP+-ASCs were expressed in the subdermal layer of the periwound margin at 10 days after injection. The GFP+-ASCs were strongly expressed and migrated into subcutaneous layers at 28 days after the local administration of ASCs. Magnification: 100x.
Analysis of GFP+-ASC populations in peripheral blood using flow cytometry. The results revealed that the percentage of circulating GFP+-ASC populations in the peripheral blood of recipients at day 28 after GFP+-ASC injection was significant compared to that in the peripheral blood of controls.
Immunofluorescent staining showed that the VEGF protein colocalized with GFP+-ASCs in the periwound area, which demonstrated that ASCs activated endothelial activity and neoangiogenic capacities after topical subdermal injection. Simultaneously, the immunofluorescent profile of von Willebrand factor (vWF) in the paraneovessel area revealed strong GFP+-ASC expression in the specimen taken from the periwound edge (Fig. 5). These results showed that ASCs induced autocrine and paracrine effects to enhance topical wound neoangiogenesis postinjection with ASCs.
Neoangiogenesis was detected in the topical periwound edge after ASC injection. (A) The immunofluorescent staining showed that VEGF expression in the periwound area apparently colocalized with GFP+-ASCs in topical subdermal areas. Double staining revealed an obvious autocrine effect of VEGF expression in GFP+-ASCs in the topical wounding edge on days 10 and 28. (B) Dual staining of specimens taken from the periwound edge revealed that GFP+-ASCs expressed vWF in the para-neovessel area and induced topical neoangiogenesis. Abbreviations: vWF, von Willebrand factor; VEGF, vascular endothelial growth factor.
XenoLight DiR-labeled ASCs were detected using an IVIS imaging system to identify ASC engraftment. The results revealed that ASCs gathered at the injection sites and migrated to the adjacent periwound edge region. Additionally, the ASCs infiltrated into the intrawound granulation tissue area after injection on day 3. The detected signals of XenoLight DiR-labeled ASCs remained strongly expressed and homed into the periwound area even at 8 weeks postimplantation (Fig. 6).
Interstitial engraftments of ASCs were detected using an IVIS imaging system. XenoLight DiR-labeled ASCs were detected. The results revealed that ASCs gathered at injection sites and immigrated into adjacent periwound edge tissues. These cells also infiltrated into the intrawound granulation tissue area after 3 days postinjection. The signals of DiR-labeled ASCs remained strongly expressed and homed into the periwound area even at 56 days postimplantation.
Discussion
Poor wound healing is a major complication in diabetes patients and can result in morbidity or death. Although experimental results have revealed promising findings, some clinical therapies have shown controversial effects (6,16). Therefore, the search for new therapeutic methods has been eagerly pursued. Previous studies have indicated that mesenchymal stem cells could aid in wound healing because of their ability to migrate into the site of injury or inflammation, to participate in the regeneration of damaged tissues, to stimulate the proliferation and differentiation of resident progenitor cells, to promote the recovery of injured cells through growth factor secretion and matrix remodeling, and to exert unique immunomodulatory and anti-inflammatory effects (18,34,38). Our previous study using a rodent dorsal wound model demonstrated the improved healing and closure of diabetic wounds following the local administration of normal BM-MSCs (25). Our results indicated that this improvement is characterized by enhanced epithelialization and by granulation tissue production. These dramatic wound healing effects may be mediated through a combination of increased fibroblast proliferation, augmented growth factor production, and enhanced cellular recruitment by promoting reepithelialization and cell infiltration. However, the preparation of BM-MSCs is time consuming. ASCs can be obtained easily and have a multilineage capacity, allowing these cells to differentiate (22,24,30). In the present study, the standard STZ-induced diabetes rodent model may not completely mimic the state of clinical patients with chronic diabetic ulcers. Elevated protease levels and long-term immune deficiencies may alter the wound bed milieu considerably in these chronic cases and can influence healing; these characteristics may not be evident in this acute rodent wound model. However, in this study, topical applications of isogeneic nondiabetic ASCs demonstrated a positive effect that enhanced diabetic wound healing.
A previous study indicated that leukocyte-mediated inflammation plays an important role in influencing the wound healing process (11). In the present study, histological analysis of the periwound tissues revealed that inflammatory cell (CD45) infiltration was attenuated in the ASC-treated group compared to that of the controls. These findings demonstrated that ASCs enhanced wound healing in association with the suppression of earlier topical proinflammatory cellular responses in the wound healing process.
Many studies have indicated that paracrine interactions that affect mesenchymal-epidermal cell-to-cell communication in the superficial epidermis and in the underlying dermis tissue are prerequisite for normal skin wound healing and are governed by the actions of certain growth factors and cytokines (37). Growth factors play an important role in the wound healing process. In the present study, IHC analysis examined cellular proliferation and regeneration in the wound margin area. Our data revealed that the ASC-treated group had a marked increase in Ki-67 expression, particularly in the fibroblasts and basal epidermal layers. EGF expression was significantly elevated in the wound margin area in the ASC-treated group, compared to that of the controls, and VEGF expression was significantly elevated in the periwound edge, particularly in the endothelial cells and fibroblasts after ASC application. These findings suggested that ASCs enhanced wound epithelialization and neovascularization via the increased expression of growth factors such as EGF and VEGF and via the induction of epithelialization in the periwound zone.
To investigate the effect of the engraftment of local applications of ASCs in cutaneous wound healing, GFP+-ASCs were injected into the periwound edge. Immunofluorescence staining indicated that many GFP+-ASCs accumulated in the subdermal layer of the wound margin at 10 days after periwound subdermal injection. The GFP+-ASCs still existed and migrated into subcutaneous layers at 28 days after the local administration of ASCs. Flow cytometric analysis of peripheral blood revealed that a higher percentage of GFP+-ASC populations could be detected in circulating blood at 4 weeks postinjection in GFP+-ASC-treated animals compared to that of the controls. These findings indicated that GFP+-ASCs not only accelerate local cell proliferation but also exert systemic effects to stimulate growth factor and cytokine expression to enhance wound healing.
In contrast, the IHC results indicated the presence of high vWF in the periwound area after the topical injection of ASCs. This finding demonstrated that topical ASC implantation could induce endothelium activation and neoangiogenesis. Our immunofluorescence study revealed that GFP+-ASCs could express both vWF and VEGF and exist in the para-neovessel area simultaneously. This evidence demonstrated that ASCs induce autocrine and paracrine effects to enhance topical wound neoangiogenesis and to promote wound healing. Furthermore, our study investigated stem cell engraftment by imaging XenoLight DiR-labeled ASCs. IVIS analysis showed that DiR-labeled ASCs were strongly expressed in the periwound edge and infiltrate to the intrawound area after injection on day 3. Interestingly, ASCs remained strongly expressed and homed into the periwound area even at 8 weeks postimplantation. This finding indicated that implanted ASCs could survive long term in the topical cutaneous area and might partially differentiate into epithelial or subcutaneous cells to enhance wound healing. Nevertheless, these data still could not provide direct evidence regarding whether ASCs can differentiate into adult somatic cells, such as fibroblasts or epithelial cells. Further studies are required to trace ASC differentiation in vivo.
The previous study revealed that the adipose-derived stromal vascular fraction (SVF) and its combination of regenerative cells have potential uses as therapeutic alternatives to increase wound healing (35). However, the therapeutic potential of a SVF isolated from diabetic mice that was administered to the wounds of diabetic mice was lower compared with cells isolated from nondiabetic mice (7). In contrast, another study indicated that autologous diabetic ASCs might be dysfunctional, particularly in their secretome (e.g., VEGF) angiogenic function, in diabetic ulcer patients (21,33). However, our study applied syngeneic nondiabetic ASCs to diabetic wounds and showed positive effects that enhanced wound healing. These results indicated that nondiabetic ASCs have a greater clinical potential application in accelerating wound healing. Further studies to compare the biomechanisms between nondiabetic ASCs and diabetic ASCs in a diabetic-induced wound model would be worthwhile.
In conclusion, the present study on a rodent wound model indicated that the topical subdermal injection of syngeneic nondiabetic ASCs into the periwound area of a diabetic wound enhances the wound healing process. ASC treatment increased tissue regeneration in association with the suppression of the topical tissue inflammatory response, augmented growth factor production, and enhanced cellular recruitment by promoting reepithelialization and cell infiltration through paracrine and autocrine mechanisms. This technique represents a feasible method for enhancing wound healing or for improving compromised tissue circulation.
Footnotes
Acknowledgments
This work was supported in part by grants Contract No. CMRPG-8B1371 and CMRPG-890351 from the Chang Gung Memorial Hospital Research Project, Taiwan. The authors declare no conflicts of interest.