Synergistic Angiogenesis Promoting Effects of Extracellular Matrix ...

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Dec 13, 2010 - be sufficient to generate a strong effect on angiogenesis.8. Endothelial cells have ...... Foundation of China (30973285). Disclosure Statement.
TISSUE ENGINEERING: Part A Volume 17, Numbers 5 and 6, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0331

Synergistic Angiogenesis Promoting Effects of Extracellular Matrix Scaffolds and Adipose-Derived Stem Cells During Wound Repair Shiyu Liu, Ph.D.,1,2,* Hongmei Zhang, Ph.D.,1,2,* Xiaojun Zhang, Ph.D.,1,2 Wei Lu, Ph.D.,1,2 Xinhui Huang, D.D.S.,3 Han Xie, D.D.S.,1 Jing Zhou, Ph.D.,1,4 Weihong Wang, D.D.S.,1 Yongjie Zhang, Ph.D.,1–3 Yuan Liu, Ph.D.,1,2 Zhihong Deng, Ph.D.,5 and Yan Jin, Ph.D.1,2

Slow vascularization rate is considered one of the main drawbacks of scaffolds used in wound healing. Several efforts, including cellular and acellular technologies, have been made to induce vascular growth in scaffolds. However, thus far, there is no established technology for inducing vascular growth. The aim of this study was to promote the vascularization capacities of scaffolds by seeding adipose-derived stem cells (ADSCs) on them and to compare the vascularization capacities of different scaffolds seeded with ADSCs. Two kinds of extracellular matrix scaffolds (small intestinal submucosa [SIS] and acellular dermal matrix [ADM]) and a kind of composite scaffold (collagen–chondroitin sulfate–hyaluronic acid [Co–CS–HA]) were selected. Subcutaneous implantation analysis showed that the vascularization capacity of SIS and ADM was greater than that of Co–CS–HA. ADSCs seeded in SIS and ADM secreted greater amounts of vascular endothelial growth factor than those seeded in Co–CS–HA. In a murine skin injury model, ADSC-seeded scaffolds enhanced the angiogenesis and wound healing rate compared with the nonseeded scaffolds. Moreover, ADSC-SIS and ADSC-ADM had greater vascularization capacity than that of ADSC-Co–CS–HA. Taken together, these results suggest that ADSCs could be used as a cell source to promote the vascularization capacities of scaffolds. The vascularization capacities of ADSC-seeded scaffolds were influenced by both the vascularization capacities of the scaffolds themselves and their effects on the angiogenic potential of ADSCs; the combination of extracellular matrix scaffolds and ADSCs exhibited synergistic angiogenesis promoting effects.

Introduction

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hree-dimensional scaffolds are presently used as dermal regeneration templates for the treatment of fullthickness skin defects resulting from injuries and severe burns. Unfortunately, the clinical benefit of these scaffolds has been poorer than expected due to their low regenerative capacity and high infection risk for the patients.1 The low vascularization, which limits the supply of immune cells, nutrients, and oxygen to the wound area, is believed to be the main reason for these problems.2 Many strategies have been employed to enhance the vascularization capacities of scaffolds. The simplest method is to incorporate angiogenic growth factors such as vascular endothelial growth factor (VEGF),3 basic fibroblast growth factor (bFGF),4 and angiogenin5; however, this method has

several drawbacks, such as the sensitivity of growth factors to thermal processing and exposure to chemical solvents,6 and the short half-life of growth factors in vivo.7 Moreover, most studies have used single growth factors, which may not be sufficient to generate a strong effect on angiogenesis.8 Endothelial cells have also been used to deal with this problem9,10; however, this procedure has not been included in clinical trials due to the difficulties in culture expansion techniques and the limited number of cells for implantation.8 Seeding stem cells, which have the ability to enhance angiogenesis in scaffolds, is being considered as a promising strategy to address this problem.2,11,12 ADSCs have been shown to differentiate into endothelial cells,13,14 enhance neovascularization in ischemic hindlimb model,15,16 and secrete angiogenic growth factors,17,18 suggesting the potential use for these cells in therapeutic vascularization and tissue

1

Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi, China. Department of Oral Histology and Pathology, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, China. 3 Engineering Technology Center for Tissue Engineering of Xi’an, Shaanxi, China. 4 Department of Pedodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, China. 5 Department of Otolaryngology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China. *These two authors contributed equally to this article. 2

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726 engineering of vascularized constructs. Moreover, ADSCs can be isolated from a small volume of adipose tissue and expanded in vitro using standard cell culture technologies.19,20 These findings indicate that ADSCs could be used as a cell source to promote the vascularization capacities of scaffolds. Extracellular matrix (ECM) scaffolds have been successfully used for wound repair in both preclinical animal studies and human clinical applications.21 The ECM is secreted by the resident cells of each tissue and organ from which it is prepared. Therefore, the composition and distribution of the ECM scaffold constituents vary depending on the tissue source. Moreover, the preparation of ECM scaffolds requires several processing steps, including decellularization, hydration, dehydration, and sterilization; these steps can affect both the structure and type of host response these scaffolds elicit when used for tissue engineering.22 Here, we selected two kinds of ECM scaffolds (small intestinal submucosa [SIS] and acellular dermal matrix [ADM]) derived from porcine, along with a composite scaffold, namely, collagen–chondroitin sulfate–hyaluronic acid (Co– CS–HA),23 consisting of Co, CS, and HA; this scaffold was developed to imitate the ingredients and their ratios in the natural dermal matrix. These scaffolds may have different vascularization capacities in vivo. Moreover, when ADSCs are seeded in these scaffolds, they not only serve as cell carriers24,25 providing mechanical support but also facilitate cell-scaffold interactions, which actively influence the cellular responses, including proliferation, differentiation,26 apoptosis,27 and angiogenic growth factor secretion by stem cells.28 Therefore, we hypothesized that the combination of ADSCs and different scaffolds would produce different effects on angiogenesis, owing to both the vascularization capacities of the different scaffolds and their influence on the angiogenic potential of ADSCs. The aim of this study was to promote the vascularization capacities of SIS, ADM, and Co–CS–HA by seeding ADSCs on them and to compare the vascularization capacities of these ADSC-seeded scaffolds by using a murine skin injury model.

Materials and Methods Preparation of scaffolds Small intestinal submucosa. SIS was developed as previously described.29 Briefly, a segment of fresh porcine jejunum was obtained from a local slaughterhouse. After carefully washing in water, the tunica mucosa, the serosa, and tunica muscularis of the segment were mechanically removed. The remaining intestinal submucosa tube was slit longitudinally and sectioned in a length of approximately 10 cm. The obtained SIS was thoroughly rinsed in a saline solution to remove the resident cells. The SIS sheets were sterilized in 0.1% peracetic acid. Finally, the resultant SIS was vacuum-sealed into hermetic packaging and stored at 48C for future use. Acellular dermal matrix. ADM was developed from porcine skin as previously described.30 Fresh porcine skin was obtained from a local slaughterhouse. After complete cleaning, excision of the subdermal fat tissue and the epi-

LIU ET AL. dermis, the resulting skin was treated with a 0.25% trypsin solution at 378C for 1.5 h and then extensively washed with distilled water. Subsequently, the dermal matrix was incubated in 1 M sodium hydroxide solution at room temperature for 16 h and thoroughly rinsed in phosphate-buffered saline (PBS) at room temperature with continuous shaking until the pH value of the entire tissue sample became neutral. ADM was lyophilized and vacuum-sealed into hermetic packaging and stored at 48C for future use. Collagen-chondroitin sulfate-hyaluronic acid. Co–CS– HA was developed as previously described.23 Bovine tendon Co I, CS, HA, 2-(N-morpholino)ethane-sulfonicacid, N-hydroxysuccinimide, and 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) were all purchased from Sigma Chemical (Badlapur). Co I was dissolved at 48C at a concentration of 12.5 mg/ml in a solution of 0.05 M acetic acid. CS and HA were dissolved at 48C at a concentration of 12.5 mg/ml in a solution of double-distilled water, respectively. The pH of Co I was adjusted to 7.4 at 48C. CS was added in Co I solution before HA was added to it. The ratio of the three elements (V/V/V) was 9:1:1. The elements were added at a speed of 0.5 ml/min. After being well mixed in culture dish with a glass rod, the slurry was poured into a 6-well plate and was frozen at –808C for 3 h and then they were lyophilized. These meshes were subsequently cross-linked for 24 h at room temperature using 40% ethanol-water (pH 5.5) solution supplemented with 50 mM 2-(N-morpholino)ethanesulfonicacid, 5 mM EDC, and 5 mM N-hydroxysuccinimide. Then, the cross-linked membranes were rinsed twice for 1 h with 0.1 M disodium phosphate, twice for 2 h with 1 M sodium chloride, six times for 24 h with 2 M sodium chloride, and ten times with double-distilled water to remove residual EDC. After being frozen again at –808C for 3 h, membranes were lyophilized and vacuum-sealed into hermetic packaging and stored at 48C for future use. Morphology examination of scaffolds Scaffolds were dehydrated by treatment with a series of grade ethanol solution (50% for 12 h, 75%, 85%, and 95% each for 2 h), and then they were placed overnight in a vacuum oven at room temperature before being coated with gold for scanning electron microscope (SEM; Hitachi S-3400N). Paraffin sections of the scaffolds were stained with hematoxylin and eosin (H&E) and visualized in a bright field using a microscope (BX-51; Olympus). Detection of nuclear remnants in scaffolds Cellular SIS (CSIS), cellular dermal matrix (CDM), and rehydrated scaffolds were embedded in tissue freezing medium optimum cutting temperature (OCT) (Leica) and immediately frozen in freezing microtome (CM1900, Leica) at 208C. Frozen sections were stained with propidium iodide (PI) (500 nM) (Sigma), and the image collection was processed by a fluorescence microscope (IX71; Olympus). Vascularization and biocompatibility of the scaffolds All the animal experiments in this study were conducted according to the committee guidelines of the Fourth Military Medical University for animal experiments, which met the

A NEW METHOD TO PROMOTE VASCULARIZATION CAPACITY OF SCAFFOLD

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2 mM insulin (Sigma-Aldrich), 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), and 10 nM dexamethasone (Sigma-Aldrich). The medium was changed every two days. After 14 days of culture, the cells were washed thrice in PBS after being fixed in 4% paraformaldehyde and then incubated in 0.3% Oil Red O (Sigma-Aldrich) solution for 15 min. After being washed thrice in PBS, cells were routinely observed and photographed under a phase-contrast inverted microscope.

NIH guidelines for the care and use of laboratory animals. Thirty-five 8-week-old C57BL/6 female mice were divided into five groups: CSIS, CDM, SIS, ADM, and Co–CS–HA. For induction of anesthesia, the mice were subsequently placed prone on the operating table and connected to a circuit delivering 3% inhalational methoxyflurane (mixed with oxygen). A constant directed flow of 1% inhalational methoxyflurane was used for maintenance anesthesia. SIS, ADM, and Co–CS–HA were sterilized by 60CO irradiation. CSIS, CDM, and these scaffolds were placed in dorsal midline subcutaneous pocked. At 7, 21, and 28 days postsurgery, the transplanted scaffolds were taken out and were immediately fixed with 4% phosphate-buffered formalin. Scaffolds transplanted for 7 and 21 days were prepared for histological analysis using H&E staining, and scaffolds transplanted for 28 days were used for immunohistochemical analysis. The primary antibodies included polyclonal rabbit anti-CD4, monoclonal rabbit anti-CD8, and monoclonal rat anti-CD68 (all from Abcam). Biotinylated secondary antibodies (Dako) were used. All the samples were examined under a microscope (BX-51; Olympus).

Osteogenic differentiation assays. A total of 2105 ADSCs were seeded into each well of a six-well plate. ADSCs were cultured in a-MEM containing 10% FBS, 0.292 mg/ml glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 5 mM L-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), and 50 mg/ml ascorbic acid. The medium was changed every two days. After 21 days of culture, cells were washed twice in PBS after being fixed in 4% paraformaldehyde and then incubated in 0.1% alizarin red solution (Sigma-Aldrich) in Tris–HCl (pH 8.3) at 378C for 30 min. After being washed twice in PBS, cells were routinely observed and photographed under an inverted microscope.

Isolation and culture of ADSCs

Preparation of stem cell-seeded scaffolds in vitro

Three-week-old C57BL/6-green fluorescent protein (GFP) transgenic mice were gifted from the Institute of Neuroscience of the Fourth Military Medical University. Adipose tissue was obtained from the inguinal region of the mice and extensively washed with 10 ml PBS. The ECM was then digested with 0.075% (w/v) collagenase type I (SigmaAldrich) at 378C for 1 h. After centrifugation, the supernatant was discarded, and the pellet was resuspended and filtered through a 100-mm cell strainer to remove undigested tissue fragments. The suspension was then centrifuged again and resuspended in 10 ml of a-minimum essential medium (MEM) (Gibco) containing 10% fetal bovine serum (FBS) (Gibco), 0.292 mg/ml glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (all from Sigma). This suspension was placed into T75 flasks and allowed to incubate at 378C in a humidified chamber containing 5% CO2 for 24 h, after which the nonadherent cells and debris were removed by aspiration. Adherent cells were then culture expanded with media changes at 2-day intervals. Cells at passage 3 were used for all experiments.

Before cell seeding, the ADM, SIS, and Co–CS–HA were sterilized by 60CO irradiation and cut into 10 mm in diameter and 0.4–0.6 mm in height. The scaffolds were individually rehydrated in culture media. Scaffolds were covered with 100 ml growth medium alone in the scaffold groups and with an equal volume of cell suspension containing 1105 ADSCs in the ADSC-scaffold groups. After 1 h of incubation, 1 ml of growth medium was added into each well. Scaffolds were incubated under standard culture conditions for 24 h, after which the overlying medium or cell suspension was aspirated. The scaffolds were flipped to place the opposite surface facing up, and the corresponding medium or cell suspension solution was placed on the other side. Scaffolds were then incubated for 24 h. The ADSC-seeded scaffolds were used for SEM, H&E staining, and transplantation.

Characterization of ADSCs Flow cytometry analysis. The phenotype of cultured ADSCs was evaluated by flow cytometry. Approximately 5105 cells were harvested by trypsin, washed twice with PBS, and incubated with phycoerythrin (PE)-conjugated rat anti-CD44, CD90, CD45 (all from Biolegend), unconjugated rat anti-CD29, CD105, and CD34 (all from Abcam), respectively. fluorescein isothiocyanate-conjugated goat anti-rat IgG secondary antibodies (Abcam) were used. Phycoerythrin (PE)-conjugated and unconjugated isotype-matching IgGs were used as controls. Cells were then analyzed on Elite ESP flow cytometry (Beckman Coulter). Adipogenic differentiation assays. A total of 2105 ADSCs were seeded into each well of a six-well plate. ADSCs were cultured in a-MEM containing 10% FBS, 0.292 mg/ml glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin,

Effect of scaffolds on the proliferation and apoptosis of ADSCs Metabolic activity assay. Metabolic activity was quantified using a 3-(4,5-dimethylthizazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS, CellTiter 96TM Aqueous; Promega) assay. ADSCs were seeded on scaffolds at a concentration of 5104 cells/cm2 in a 96-well plate. At 1, 3, 5, and 7 days, 100 ml of FBS-free culture medium and 20 ml of MTS reagent were added into every specimen. After incubation for 2 h in a CO2 incubator, all the reactant mixtures were extracted and added into 96-well plates. Then, the absorbance of each specimen in the 96-well plates was measured in a micro-plate reader at a wave length of 490 nm. Background absorbance was corrected by subtracting the absorbance index of culture medium from the specimen data. Flow cytometry analysis. After being seeded onto SIS, ADM, and Co–CS–HA, respectively, at a concentration of 2104 cells/cm2 and cultured for 72 h in a 24-well plate, cells were trypsinized as previously described; 31 and cell precipitates were washed twice with PBS and resuspended in 1 ml of physiological saline by repeated vibration to ensure a

728 single-cell suspension. Then, 2 ml of cold dehydrated alcohol was quickly mixed with the cell suspension to fix cells at 48C for 24 h. Finally, the cells were washed twice again with PBS, stained with 100 mg/ml propidium iodide at 48C for 30 min, and subjected to cell-cycle analysis using Elite ESP flow cytometry (Beckman Coulter). For apoptosis analysis, singlecell suspension of ADSCs was washed twice with cold PBS, incubated for 15 min with fluorescein-conjugated annexin V and PI, and analyzed using the same flow cytometer. Effect of scaffolds on the angiogenic growth factors secretion by ADSCs Real-time polymerase chain reaction analysis. After being seeded onto SIS, ADM, and Co–CS–HA, respectively, at a concentration of 2104 cells/cm2 and cultured for 72 h in a 24-well plate, ADSCs were trypsinized and were used for RNA extraction. Total cellular RNA was exacted by using the TRIzol Reagent (Invitrogen Life Technology), and first-strand cDNA synthesis was performed according to the manufacturer’s protocol. Real-time polymerase chain reaction products were detected with SYBR Green dye by using Light Cycler Instrument (Toyobo). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) gene was amplified as internal control. Primer sequences were as follows: VEGF: 50 -primer (50 -AGAGCAACATCACCATGCAG-30 ) and 30 primer (50 -CAGTGAACGCTCCAGGATTT-30 ); hepatocyte growth factor (HGF): 50 -primer (50 -GTGGATGCCAAGC CAAGCT-30 ) and 30 -primer (50 -CAGTAGGGTGGATGGTTA GTTTGAA-30 ); bFGF: 50 -primer (50 -CACCAGGCCACTTC AAGGA-30 ) and 30 -primer (50 -GATGGATGCGCAGGAAG AA-30 ); GAPDH: 50 -primer (50 -ATCATCCCTGCATCCACT30 ) and 30 -primer (50 -ATCCACGACGGACACATT-30 ). We calculated expression levels by the comparative CT method using GAPDH as an endogenous reference gene. Enzyme-linked immunosorbent assay analysis. SIS, ADM, and Co–CS–HA were seeded with ADSCs, respectively, at a concentration of 2104 cells/cm2 and cultured in a 24-well plate for 6 days. Scaffolds without ADSCs were also cultured as control. Every 48 h, the conditioned medium of ADSC-seeded scaffolds and empty scaffolds was collected, respectively, and assayed using commercially available sandwich enzyme-linked immunosorbent assays (R&D Systems) for mouse VEGF. Then, fresh medium was added. At the same time, ADSCs were trypsinized, and cell counts were performed with a hemacytometer. VEGF levels were normalized to the number of cells at the time of harvest, expressed as pg per 106 cells. Full-thickness cutaneous wound model A total of forty-nine 8-week-old female C57BL/6 mice were randomized to seven treatment groups: control, only scaffolds implantation (3 groups), and scaffolds seeded with ADSCs (3 groups). Each mouse was anesthetized as previously described, and a 1-cm diameter punch biopsy instrument was placed with moderate force onto the dorsum of the mouse to create an impression of the circumference. Next, the middle of the outlined region of skin was grasped with forceps, and the 1 cm diameter region was sharply excised along the outline with a pair of scissors. The excised tissue was full-thickness skin in depth, leaving subcutaneous dorsal muscle exposed

LIU ET AL. after excision. In the scaffold and ADSC-scaffold groups, the grafts were removed from PBS and were placed into the dorsal wound. The wound was then covered by two layers of vaseline gauze with discontinuous suture onto the marginal recipient mice skin of the defect area by 3-0 silk suture. In the control group, the wound was just covered by two layers of vaseline gauze and discontinuously sutured. Wound healing rate To determine the rate of wound healing in wound area, the wounds were imaged by digital camera after surgery at 0–3 weeks. Photographs were uploaded to an appropriate computer platform and were analyzed using the Image J image analysis software (NIH image software). All photographs were taken with the experimental mouse placed adjacent to a metric ruler that was used for distance calibration and standardization, allowing subsequent quantitative analysis. The percentage of wound closure was calculated as follows: (area of original wound–area of actual wound)/area of original wound100%. Gross study of wound bed cutaneous vascular infiltration Animals were euthanized at postoperative day 21, and approximately 33 cm, full-thickness cutaneous biopsies of the wound repair bed and surrounding tissue were obtained. Tissue specimens were carefully placed on the bottom of a polystyrene cell culture dish (Corning Enterprises) and spread with moderate tension along the plate bottom, with the superficial surface facing up. Next, standard incandescent illumination was immediately directed under the specimen into the polystyrene dish, illuminating the vascular infiltration of wound bed biopsy specimens. Photographs were taken with a digital camera. After visualization of the vessels, tissue specimens were divided into two parts. One part was placed in 4% phosphate-buffered formalin and prepared for staining with H&E. The second part was embedded in tissue freezing medium optimum cutting temperature (OCT) (Leica) and immediately frozen in freezing microtome (CM1900; Leica) at 208C to investigate the immunofluorescent analysis. Immunofluorescent analysis and measurement of microvessel density GFP-positive ADSCs were identified with a polyclonal rabbit-anti-GFP primary antibody (Abcam); endothelial cells were identified with a monoclonal rat-anti-CD31 primary antibody (Abcam). Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Invitrogen) and Fluor 594 goat anti-rat IgG secondary antibody (Invitrogen) were used. Hoechst 33342 dye was used to stain nuclei. The signals were examined under a laser scanning confocal microscope (FV-1000; Olympus). The number of microvessels was counted. In each section, three high-power fields were randomly selected and photographed, and microvessels were counted in each field. Vessels with a diameter of