Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing

Acta Biomaterialia 69 (2018) 156–169

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Highly efficient local delivery of endothelial progenitor cells significantly potentiates angiogenesis and full-thickness wound healing Chenggui Wang a, Qingqing Wang a, Wendong Gao a,b, Zengjie Zhang a, Yiting Lou a, Haiming Jin a, Xiaofeng Chen b, Bo Lei c, Huazi Xu a,⇑, Cong Mao a,b,⇑ a Key Laboratory of Orthopedics of Zhejiang Province, Department of Orthopedics, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China b National Engineering Research Center for Tissues Restoration and Reconstruction, South China University of Technology, Guangzhou 510640, China c Frontier Institute of Science and Technology, State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710000, China

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Article history: Received 17 August 2017 Received in revised form 19 December 2017 Accepted 16 January 2018 Available online 1 February 2018 Keywords: Bioactive glass Cell-delivery nanocomposites Angiogenesis Endothelial progenitor cells Wound healing

a b s t r a c t Wound therapy with a rapid healing performance remains a critical clinical challenge. Cellular delivery is considered to be a promising approach to improve the efficiency of healing, yet problems such as compromised cell viability and functionality arise due to the inefficient delivery. Here, we report the efficient delivery of endothelial progenitor cells (EPCs) with a bioactive nanofibrous scaffold (composed of collagen and polycaprolactone and bioactive glass nanoparticles, CPB) for enhancing wound healing. Under the stimulation of CPB nanofibrous system, the viability and angiogenic ability of EPCs were significantly enhanced through the activation of Hif-1a/VEGF/SDF-1a signaling. In vivo, CPB/EPC constructs significantly enhanced the formation of high-density blood vessels by greatly upregulating the expressions of Hif-1a, VEGF, and SDF-1a. Moreover, owing to the increased local delivery of cells and fast neovascularization within the wound site, cell proliferative activity, granulation tissue formation, and collagen synthesis and deposition were greatly promoted by CPB/EPC constructs resulting in rapid reepithelialization and regeneration of skin appendages. As a result, the synergistic enhancement of wound healing was observed from CPB/EPC constructs, which suggests the highly efficient delivery of EPCs. CPB/ EPC constructs may become highly competitive cell-based therapeutic products for efficient impaired wound healing application. This study may also provide a novel strategy to develop bioactive cell therapy constructs for angiogenesis-related regenerative medicine. Statement of Significance This paper reported a highly efficient local delivery of EPCs using bioactive glass-based CPB nanofibrous scaffold for enhancing angiogenesis and wound regeneration. In vitro study showed that CPB can promote the proliferation, migration, and tube formation of EPCs through upregulation of the Hif-1a/VEGF/SDF-1a signaling pathway, indicating that the bioactivity and angiogenic ability of EPCs can be highly maintained and promoted by the CPB scaffold. Moreover, CPB/EPC constructs effectively stimulated the regeneration of diabetic wounds with satisfactory vascularization and better healing outcomes in a full-thickness wound model, suggesting that the highly efficient delivery of EPCs to wound site facilitates angiogenesis and further leads to wound healing. The high angiogenic capacity and excellent healing ability make CPB/EPC constructs highly competitive in cell-based therapeutic products for efficient wound repair application. Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding authors at: Department of Orthopedics, The Second Affiliated Hospital of Wenzhou Medical University, NO.109 XueYuan West Road, Wenzhou 325000, China. E-mail addresses: [email protected] (H. Xu), [email protected] (C. Mao). https://doi.org/10.1016/j.actbio.2018.01.019 1742-7061/Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Skin wound caused by trauma, burns, and chronic diseases remains a great clinical challenge worldwide [1,2]. In general, wound healing is a complex process that is classically divided into three stages including inflammation, proliferation, and remodeling,

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which requires the efforts of multiple cells, growth factors, and extracellular signals [3]. However, owing to several reasons such as ischemia, diabetes, and pressure, chronic wounds usually occur in combination with complications of continuous inflammation, cell dysfunction, and impaired angiogenesis, leading to long-term medical care, high cost, and compromised quality of life in patients with these diseases [4]. The conventional treatment for wounds would be surgical debridement and assistant wound care methods including antibiotics and wound dressings [5]. However, standard surgical treatment with skin substitutes such as autograft, xenograft, or tissue engineered skin has its own disadvantages including limited availability, secondary surgeries, high cost, delayed healing, and fibrosis problem [6]. To overcome these difficulties, cell therapy can be a potential alteration for wound healing [7,8]. Stem cells such as mesenchymal stem cells, endothelial progenitor cells, and epithelial stem cells have been investigated for wound healing applications because of their ability to secrete bioactive factors that can enhance granulation tissue formation, angiogenesis, and reduce inflammation [9–12]. Although problems such as poor engraftment and high rates of cell death often compromise the efficiency of cell-based therapies, stem cells remain as promising approaches for wound healing as they can integrate environmental signals and transduce them into biological factors in wound bed [13]. The endothelial progenitor cell (EPC) is a type of bone marrow mononuclear progenitor cell that acts as endothelial precursor that increase angiogenesis and vascularization by secreting growth factors and cytokines in damaged tissues [14]. EPCs play an important role in vascular regeneration when compared with other stem cells [15]; this is associated with their characteristics such as differentiation into vascular endothelial cells (ECs) and their existence in the vascular wall [16]. Transplantation of EPCs has demonstrated promising results in wound healing [17]. However, the mode of EPC transplantation, such as in situ injection or intravenous injection, cannot achieve the on-site local delivery and will compromise the survival and function of EPCs [8,18]. Therefore, it will be of great significance to fabricate an ideal skin tissue engineering scaffold that is highly bioactive and can provide a temporary residence for maintaining the viability and activating the angiogenic function of EPCs. To achieve this goal, electrospinning nanofibrous scaffolds with good biocompatibility and high bioactivity can be used as an efficient therapeutic approach for the delivery of EPCs into the wound site [19]. For skin tissue engineering, collagen (Col) fibers are commonly used because they are the main constituent of skin extracellular matrix (ECM), which could create a biomimetic microenvironment for cells to attach and proliferate during wound healing [20]. However, owing to their poor mechanical properties and rapid degradation, collagen fibers should be combined with other materials to obtain a stable and useful scaffold. Various polymer-blended electrospun fibers such as Col/polycaprolactone (PCL), chitosan/PCL, polylactic-co-glycolic acid (PLGA)/Col have been examined for promoting wound healing, yet results are still unsatisfactory owing to their compromised bioactivities and delayed angiogenesis [21–23]. Bioactive glasses (BGs) have shown successful applications both in hard and soft tissue repair because of their high biocompatibility and bioactivity [24–26]. Recently, bioactive glass nanoparticles (BGNs) featured with regular size, large specific surface area, and high bioactivity have presented enhanced cell responses by releasing Si, Ca, and P ions [27–30]. Previous studies showed that BGNs can stimulate the angiogenic ability of human umbilical vein endothelial cells (HUVECs) by upregulating specific angiogenic growth factors such as VEGF and bFGF [31,32] and can efficiently enhance wound healing [27,28]. Moreover, mature bone ECM composed of inorganic apatite nanocrystals and collagen nanofibers has shown a strong capacity for angiogenesis by efficiently mediating cellular differentiation

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and secreting growth factor [33]. Therefore, it is reasonable to speculate that a BGN-based collagen nanofibrous structure could enhance the delivery efficiency of EPCs and accelerate wound healing. In this study, we investigated the local delivery performance of EPCs and the mechanism of Col-PCL-BGN (CPB) nanofibrous scaffolds in wound healing. It is hypothesized that the combination of BGNs and collagen could exert a synergistic positive effect as a cell-delivery system on maintaining the viability and activating the angiogenic ability of EPCs and therefore could accelerate wound healing. The regulation and mechanism of CPB scaffolds on EPC behaviors in vitro and wound healing efficiency of the CPB/EPC delivery system in vivo were investigated.

2. Materials and methods 2.1. Characterizations of CPB-EPC constructs The fabrication and characterization of the CPB and CP nanofibrous scaffolds were described in our previous study [34]. The bone marrow-derived EPCs from Sprague Dawley (SD) rats were isolated as described in Supporting Information, and after 1 week of culture, EPCs were characterized by DiI-Ac-LDL/FITC-UEA staining, immunostaining of CD31 and KDR, and the tube formation assay. All EPCs used in this study were cultured in EGM-2MV and in passages 3–5. The CPB/EPC and CP/EPC constructs were obtained by seeding 2
104 EPCs on CPB and CP scaffolds and cultured for 24 h. To evaluate the biocompatibility of CPB nanofibrous scaffolds, cell attachment and proliferation morphology were analyzed by SEM and laser scanning confocal microscopy (Olympus, BX61W1FV1000, Japan). For SEM observation, specimens were fixed using 4% paraformaldehyde for 30 min after 48 h of culture and subsequently dehydrated in increasing concentrations of ethanol (15%-–100% v/v). After drying, the specimens were mounted and coated with gold and observed by SEM (Hitachi H-7500, Japan) at an accelerating voltage of 15 kV. For laser scanning confocal microscopy, F-actin and cell nucleus were stained by phalloidin-FITC and 40 -6-diamidino-2-phenylindole (DAPI), respectively. Briefly, samples were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 in phosphate buffered saline (PBS) for 10 min, and then incubated with phalloidin-FITC (1:500; Cytoskeleton, Acoma St., Denver, USA) in dark for 1 h. After washing, cell nuclei were stained with DAPI. Slides were maintained in a humidified dark box at 4 °C before observation. The proliferation of EPCs on different nanofibrous scaffolds was measured by the Cell Counting Kit-8 (CCK-8, Dojindo). Briefly, 100 lL of suspended EPCs at a density of 1
105 cells/ml were seeded on the CPB and CP scaffolds, which were previously placed in a 24well plate (Corning), while cells seeded on the blank cell culture cover slips (Solarbio) were used as the control. After 1 day, 2 days, and 3 days of culture, samples were washed using PBS and added with 200 lL of medium supplemented with 20 lL of CCK-8 reagent, followed by incubating for 1 h. The mixed medium was then transferred into a 96-well plate, and the absorbance was measured at 450 nm wavelength using a microplate reader (Multiskan GO; Thermo Scientific). A tube formation assay with growth factor-reduced Matrigel (BD Biosciences, US) was also performed to evaluate the effects of CPB scaffolds on angiogenic morphogenesis and tube formation capacity of EPCs. Briefly, the Matrigel solution was thawed at 4 °C overnight and then placed in a l-Slide (10 lL per well; IBIDI, Germany) in a cell incubator for 30 min for solidification. After coculturing with CP/CPB for 48 h, a total of 5000 cells per well were detached from the scaffolds using trypsin for 3 min and then seeded in the Matrigel-precoated l-Slide. EPCs detached from cell

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culture slides were used as the control. After 4 h of incubation, tube formation was observed and quantified; five independent fields were imaged, and tube circles and nodes were counted for an average number under an inverted light microscope (Nikon).

CP/EPCs, and control were harvested and embedded for staining by DAPI. A total of 20 different granulation tissue fields (5 fields for each section and 4 sections from each group) were selected in each group, and the percentages of DiI-labeled EPCs were calculated.

2.2. Cellular immunofluorescence assay 2.5. Assessment of blood flow in the wound area EPCs were seeded on the nanofibrous scaffolds as described above. After 48 h of culture, samples were fixed with 4% paraformaldehyde, permeabilized in PBS containing Triton X-100 for 10 min, and then were blocked with 10% bovine serum albumin at 37 °C for 1 h, followed by incubating with primary antibody: mouse monoclonal anti-CD31 (ab64543, 1:400; Abcam), rabbit polyclonal anti-KDR (ab2349, 1:300; Abcam), rabbit monoclonal anti-VE-cadherin (ab205336, 1:200; Abcam), rabbit polyclonal anti-HIF-1a (NB100-105, 1:200; Novus, US), or without primary antibody but with PBS (as negative controls) at 4 °C overnight, respectively. Then, the slides were washed and incubated with fluorescein isothiocyanateor tetramethyl rhodamine isothiocyanate-conjugated secondary antibody for 1 h at 37 °C. Finally, cell nuclei were visualized by DAPI (Beyotime Institute of Biotechnology), and cytoskeletons were visualized by F-actin and viewed under confocal microscope system (BX61W1-FV1000; Olympus, Japan). Three fields of view were randomly selected for observation and quantification on each slide with a fluorescence microscope (Olympus Inc., Tokyo, Japan), and the staining intensity was measured using Image-Pro Plus 6.0 by observers who were blinded to the experimental groups. 2.3. Animal model of full-thickness wounds The CPB/EPC and CP/EPC constructs were prepared by the methods as described in Section 2.1. All animals were ordered from the SLAC laboratory animal company in Shanghai, China, and all animal procedures were approved by the Wenzhou Medical University Animal Care and Use Committee (approved license number: wydw2016-0157). For wound model, a total of 35 male SD rats (220–250 g) were anesthetized with 2.5% pentobarbital sodium (30 mg/kg) and randomly selected for grouping. After shaving and sterilization, two full-thickness wounds (20 mm in diameter) were made by scissor cut along the mark on each side of the rat’s back. Vehicle alone, EPC-free CP/CPB scaffolds, or EPC-seeded CP/CPB scaffolds were used to cover the wounds (n = 7 animals per group), and the cell-loaded scaffolds were placed facing down with cells directly contacting the wound sites. All treated wounds including those in the control group were then applied a TegadermTM transparent dressing (3M Health Care, Germany). All the animals were fed in individual cages and given ad libitum feeding access to food and water, and they were observed every day during the total period of the experiment. At days 0, 3, 7, 14, and 21 posttreatments, the wound area was calculated by tracing the wound margins from rats and was evaluated as a percent area of the original wound using Image-Pro Plus 6.0 software. 2.4. Transplantation of DiI-Ac-LDL labeled EPCs Another six rats were used to evaluate the aggregation of locally delivered EPCs at the wound site. Briefly, cells were labeled with 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-Ac-LDL; Sigma, MO, USA) for 24 h, as previously described [35]. The DiI-Ac-LDL labeled 2
104 EPCs were seeded on CPB and CP scaffolds for 24 h to obtain the CPB/EPC and CP/EPC constructs, respectively, which were then transplanted into the wounds of the rats with EPCs directly contacting with the wounds. Local injection of 2
104 EPCs was set as the control. After 7 days post-surgery, wounds of CPB/EPCs,

The blood flow in the wound area was assessed by a laser Doppler imager (MoorLDI-2; Moor Instruments Limited, Devon, UK) with a laser wavelength of 633 nm, scan of 55 cm, and scan duration of 5 min at 25 ± 5 °C. Briefly, the rats were anesthetized with 2.5% pentobarbital sodium (30 mg/kg), shaved, and then gently fixed onto a black platform. MoorLDI Review V6.1 software was used to quantify the results. 2.6. Histological analysis Animals were anesthetized with 2.5% pentobarbital sodium (30 mg/kg) and killed by injection with 8% (w/v) chloralic hydras after skin dissection at specific time points. For Hematoxylin and Eosin (H&E) staining, Masson trichrome staining, and immunohistochemistry, 0.5 cm section of the skin was dissected out, postfixed by 4% paraformaldehyde for 6 h, and then embedded in paraffin. Sections (5 mm thick) were mounted on slides for following staining. For H&E (Beyotime Institute of Biotechnology, China) staining, slides were stained with H&E to visualize tissue formation following the manufacturer’s instruction. Masson trichrome staining was also performed for observation of collagen deposition during the healing period. Images were captured and analyzed using a Nikon ECLPSE 80i (Nikon, Japan). 2.7. Immunohistochemical and immunofluorescence staining Sections mounted on slides were deparaffinized and rehydrated and then immersed in 3% H2O2 and 80% carbinol for 15 min at room temperature to block the endogenous peroxidase activity. To recover the antigen, slides were immersed into 10 mM citrate buffer and heated in a microwave oven twice. Non-specific binding sites were blocked with 5% bovine serum albumin (BSA) (Beyotime) in PBS for 1 h at room temperature. The primary antibody of rabbit polyclonal anti-Ki67 (ab15580, 1:200; Abcam), rabbit polyclonal anti-collagen III (ab7778, 1:300; Abcam), or rabbit polyclonal anti-collagen I (ab21286, 1:300; Abcam) was incubated with tissue at 4 °C overnight. After washing, slides were then added with HRP goat anti-rabbit antibody (1:400; Abcam) for 1 h. The antibody-binding sites were visualized by incubation with a DAB chromogen kit (ZSGB-BIO; Beijing, China), then counterstained with hematoxylin for 5 min. All slices were observed with a Zeiss 40FL Axioskop fluorescent microscope. The day-7 skin sections were stained with rabbit polyclonal anti-cytokeratin (ab9377, 1:75; Abcam), anti-Hif-1a (NB100-105, 1:200; Novus), rabbit polyclonal anti-a-SMA, or without primary antibody but with PBS (as negative controls), followed by incubation with goat anti-rabbit IgG Alexa FluorÒ 488/647-conjugated secondary antibody (ab150077/ab150083, 1:1500; Abcam), and nuclei were stained by DAPI (Beyotime). All fluorescent images were taken using a Nikon confocal laser microscope (A1 PLUS; Nikon, Tokyo, Japan). 2.8. Western blot analysis The EPCs co-cultured with scaffolds for 3 days or the wound tissues were completely homogenized in RIPA lysis buffer or a tissue grinding machine. The extracts were first quantified with BCA reagents. Then, samples containing 30 lg (cells) or 80 lg (tissue

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Fig. 1. Biocompatibility of CPB nanofibrous scaffolds. (A) SEM images showing morphology of EPCs on scaffolds at day 2 (Scale bar: 20 lm); (B) cell morphology and F-actin arrangement of EPCs on scaffolds at day 2 (Scale bar: 10 lm); (C) cell proliferation during culture of 1–3 days; (D) in vitro tube formation assay of EPCs induced by CPB (Scale bar: 100 lm); (E) quantification of tube number; n = 3 independent experiments.

sample) protein were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 2 h and incubated at 4 °C overnight with primary antibodies as follows: anti-Hif-1a (1:500, NB100-105; Novus, US); antiVEGF (1:1000, NB100-664; Novus, US); anti-SDF-1a (1:1000,

AF5166; Affinity, US), after which they were incubated with horseradish peroxidase–conjugated secondary antibodies for 2 h. The bands were detected by electrochemiluminescence reagent (Invitrogen), and the signals were visualized by a ChemiDocXRS+ Imaging System (Bio-Rad).

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Fig. 2. CPB scaffolds activate the HIF-1a/VEGF/SDF-1a signaling pathway in EPCs. (A) Western blot analysis and quantification of Hif-1a, VEGF, and SDF-1a proteins in EPCs cultured on scaffolds for 3 days; (B) fluorescent images of F-actin (red) and Hif-1a (green) in EPCs cultured on scaffolds for 3 days; (C) quantification of the cellular immunofluorescence intensity of Hif-1a; Scale bar are 20 lm, n = 3 independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.9. Statistical analysis Data of at least three individual experiments are presented as mean ± standard deviation (mean ± SD). Before applying a parametric or non-parametric test to assess differences between groups, the normal distribution of all variables was tested. If all variables met the normal distribution, the statistical trends were analyzed by the one-way ANOVA with post-test using the Tukey’s test; otherwise, the Kruskal–Wallis test (Graph Pad Prism 7.0) was used. Statistical significance was set at *P < 0.05 and **P < 0.01 versus the indicated group.

3. Results and discussion 3.1. Characterization of CPB-EPC constructs Rat EPCs were successfully isolated from the femurs and tibias of 2-week-old SD rats. In the initial week, proliferative endothelial colonies which are indicative of BM-EPCs were observed (Fig. S1A). In contrast, EPCs cultured for over 2 weeks showed a cobblestonelike morphology and lacked cluster-like formations. Strong expression of BM-EPC markers (CD31 and KDR) confirmed their

EC characteristics (Fig. S1B). After co-culturing with DiI-Ac-LDL, positive staining for DiI-Ac-LDL and UEA lectin was observed in BM-EPCs (Fig. S1C). The tube formation assay results revealed their angiogenic ability in vitro (Fig. S1D). These results indicated that cells derived from bone marrow (passages 3 to –5) were predominantly EPCs and maintained both endothelial characteristics and differentiation potential. Fig. 1 shows the cellular biocompatibility of CPB scaffolds toward EPCs. In Fig. 1A, cells on CP and CPB nanofibrous scaffolds showed favorable initial adhesion and spreading, which both exhibited with flattening morphology. In addition, the TRITCPhalloidin staining presented that cells had a cobblestone-like morphology and well organized actin stress on the CPB group compared with the CP and control (Ctr) groups (Fig. 1B), indicating a proper proliferation status of EPCs. As shown in Fig. 1C, a CCK-8 method was used to quantify the cytotoxicity of CP/CPB scaffolds. Results indicated that both scaffolds supported the growth of EPCs, and no significant cytotoxicity of the scaffold to the cells was found. Moreover, compared with the control group, the nanofibrous scaffolds enhanced the proliferative ability of EPCs in a time-dependent manner. All these results indicated that the CPB scaffolds showed good biocompatibility and could promote proliferation of EPCs.

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Fig. 3. CPB/EPC constructs accelerate wound closure. (A) Double immunofluorescence staining of DiI-EPCs and a-SMA of the regenerated tissue. Red fluorescence indicates locally delivered EPCs, green fluorescence indicates a-SMA around capillaries, and double-positive cells indicate locally delivered EPCs differentiated into endothelial cells and formed vessels. Scale bar: 100 lm. (B) Quantification of the DiI-positive cells and newly formed vessels; n = 4 per group. (C) Representative images of healing in wounds treated with saline control, CP scaffold, CPB scaffold, CP/EPC construct, and CPB/EPC construct at day 0, 3, 7, 14, and 21; (D) The wound closure rates of all five groups; # means P < 0.05 of CPB/EPC versus CPB groups; n = 4 per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. In vitro angiogenesis and mechanism of EPCs mediated by CPB scaffolds The ability of CP/CPB scaffolds to promote tube formation of EPCs was assessed using the tube formation assay. As shown in Fig. 1D, tube-like structures were formed in all three groups after 4 h of culture. However, the tubes in nanofibrous scaffold groups exhibited more mature tubes with nodes, circles, and tube structures with integrity. Specifically, the CPB scaffolds notably stimulated the formation of vascular tubes in EPCs compared to CP scaffolds and control group (Fig. 1E), indicating that the tube formation ability of EPCs was significantly enhanced by the CPB scaffolds. The levels of the angiogenic proteins CD31, KDR, and VE-Cadherin, which are expressed in normal vascular development related to wound healing process {Carmeliet, 2000 #314} [36–39], were detected by immunofluorescence staining in EPCs (Fig. S2). A large amount of positive staining of CD31, KDR, and VE-Cadherin was observed in the CPB and CP groups, while less activated proteins were found in the control group (Fig. S2A, C, E). Additionally, the relative fluorescence intensities of the three proteins in CPB scaffolds were statistically significantly higher than those of the CP and control groups (Fig. S2B, D, F). These results suggested that CPB scaffolds can activate and upregulate angiogenic-related protein expression, which further enhanced the tube formation ability of EPCs in vitro (Fig. 1D). To better understand the mechanism of CPB scaffolds that promote angiogenesis, the effect of nanofibrous scaffolds on hypoxiainducible factor-1 (Hif-1a), a crucial physiological regulator of vascularization, and its two downstream genes, VEGF and SDF-1a [40], was evaluated in this study. It was shown that the protein levels of Hif-1a were significantly upregulated by the use of nanofibrous scaffolds when compared with control, and CPB scaffolds exhibited higher expression than the CP group. The expressions of the two Hif-1a-targeted genes, VEGF and SDF-1a, which are essential for angiogenesis, followed a similar changing pattern when co-cultured with the scaffolds and showed the highest protein levels in the CPB group (Fig. 2A). The results of immunofluorescence of the activated Hif-1a also confirmed the aforementioned western blot analysis and suggested that the amount and distribution of the Hif-1a protein was dependent on the type of substrate. For cells seeded on CPB scaffolds, the acti-

vated Hif-1a distributed mostly in the cell nucleus region, characterized by large amount of round dots and significantly higher immunofluorescence intensity than the CP and control groups (Fig. 2B, C). These results revealed that CPB scaffolds can stimulate the angiogenic ability of EPCs through the Hif-1a/VEGF/SDF-1a cascade signaling. 3.3. Aggregation of locally delivered EPCs and wound healing On the basis of the above results, CPB scaffolds enhanced angiogenesis of EPCs in vitro, which may facilitate cutaneous wound healing. Because EPC transplantation induced angiogenesis and enhanced healing in the ischemic tissues [17,41,42], the local injection of EPCs was given in the present study to evaluate the ability of efficient aggregation of EPCs at the wound site in comparison to CPB/EPC tissue constructs (Control group in Fig. 3A). It is clearly shown from tissue immunofluorescence staining that the CPB/ EPC group displayed statistically significant stronger DiI-labeled positive staining with more DiI-positive EPCs than CP/EPCs and local injection of EPCs (control), indicating that EPCs can migrate from the CPB/EPCs constructs to the granulation tissue right below the scar and aggregate more cells in the wound area than the local injection of EPCs or the CP/EPC scaffold (Fig. 3B). With these locally delivered EPCs, angiogenesis and functional blood supply in wound tissue will be quickly achieved, eventually leading to better healing results [43,44]. Moreover, we performed co-staining of DiI-EPCs and a-SMA of the wound tissue after 7 days of transplantation. Results showed that the CPB/EPC group formed more vessels (22. 67 ± 3.05), which was statistically significant than CP/EPC and local injection of EPCs (control), indicating that the locally delivered EPCs contributed to the development of newly formed blood vessels and the migrated cells maintained high viability and could efficiently differentiate into ECs to form blood vessels. Therefore, CPB/EPC tissue constructs should be a great candidate for wound healing, and the wound healing effects of CPB scaffolds containing EPCs were investigated in full-thickness cutaneous wounds in rats, in comparison to saline, CP scaffolds with or without EPCs as controls. During the experimental period, no death or severe inflammation was found in all rats. As is clearly shown by the gross observation images in Fig. 3C, all wounds treated by the nanofibrous

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Fig. 4. Histology evaluation of wounds treated by CPB/EPC constructs. (A) H&E staining images of full-thickness wounds on days 7 and 21; the black inverted triangle area represents the epidermis (except for wound area), arrows indicate newly formed dermal appendages, scale bar: 100 lm; (B) quantification of the length of the wound site at days 7; (C) quantification of the thickness of the granulation tissue on day 7; (D) quantification of the length of the wound area on day 21; # P < 0.05 of CPB/EPC versus CPB group; n = 4 per group.

scaffolds with or without EPCs (CP, CPB, CP/EPC, and CPB/EPC groups) achieved obvious reduction in wound area when compared with control. Wound closure was significantly promoted by CPB/EPC constructs during the whole healing process. Consistent with the gross inspection results, CPB/EPC constructs displayed the highest quantitative wound healing rates at all time points among all the five groups. Specifically, although no difference was found among the five groups during the first 3 days post-op, wound healing rate was statistically greatly accelerated by CPB/EPC constructs on day 7. On day 14, the healing rates in all groups slowed; however, the CPB/EPC construct group still achieved the highest rate compared to others. By day 21, wounds in the CPB/EPC scaffold group was completely closed with a healing rate of 100%, while other wounds remained unhealed with significantly lower healing rates (Fig. 3D), demonstrating that CPB/EPC constructs accelerated wound healing faster than the CP/EPC group in vivo. These results demonstrate the potent therapeutic function of CPB/EPC constructs on wound healing. In addition, data on wound closure showed that the CPB/EPC group possessed statistically significant better healing rate than the CP/EPC and CPB groups, indicating that CPB and EPCs may synergistically promote would healing.

3.4. Accelerated granulation formation and re-epithelialization To further assess the healing quality of wounds treated by CPB/ EPC constructs, H&E staining was performed on tissue samples on the wound site. The granulation tissue, which consists of large numbers of cells, cytokines, blood vessels, and extracellular matrix, begins to fill the wound space in about 4 days after injury [2]. As shown in Fig. 4A, neoepidermis can already be found in the treated wounds of CPB/EPC and CP/EPC constructs, while wounds in other three groups showed no noticeable neoepidermis at day 7. Consistent with the observation results of H&E, the CPB/EPC constructs showed the shortest wound length among all the groups, followed by CP/EPC, CPB, CP, and control groups (Fig. 4B). Meanwhile, the thickness of granulation tissue appeared to be of significantly great difference among all the five groups, characterized with continuous layers of granulation across the entire wound gap in the CPB/ EPC and CP/EPC groups but much thinner regenerated tissue in the wounds of the control group. Still, the wounds treated only with nanofibrous scaffolds exhibited thicker granulation tissue than the control group (Fig. 4C). At day 21, all wounds except those in the control group closed and were covered with neoepidermis. However, wounds treated by CPB and CP scaffolds remained

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Fig. 5. Immunostaining of cytokeratin expression in wounds treated by CPB/EPC constructs. (A) Fluorescence images of wound sections stained with cytokeratin at day 7, scale bar: 100 lm; (B) the close-up images of cytokeratin-positive area in A, scale bar: 20 lm; (C) quantification of the length of wound area on day 7; (D) Quantification of the thickness of epithelial cells on day 7; # P < 0.05 of CPB/EPC versus CPB groups; n = 4 per group.

unhealed with an open length of 0.49 ± 0.06 and 0.73 ± 0.04 cm, respectively, which were still significantly shorter than the ones in the control group (Fig. 4D). Surprisingly, the treated wounds in the CPB/EPC group showed a mature epithelial structure and newly formed dermal appendages in the scar tissue, while none was found in the other groups. Normally, wound healing in adult mammals leads to a scar tissue that is lack of skin appendages [45]. These results demonstrated that CPB/EPC constructs can not only accelerate wound healing but also facilitate the formation of mature epithelium and skin appendages, showing their great potential in complete wound repair and regeneration. Cytokeratin is a crucial marker in evaluating epidermal differentiation and re-epithelialization of wound healing [46]. Fig. 5 shows the cytokeratin immunostaining results of the regenerated tissue at day 7 corresponding to different treatments. The CPB/ EPC and CP/EPC groups showed rapid re-epithelialization than scaffold alone and control groups, as confirmed by shorter wound length and stronger positive staining of cytokeratin within the neoepidermis. Moreover, the neoepidermis, which was the thickest in the treated wounds of scaffold/EPC constructs (Fig. 5A, B), exhibited a differentiated structure with more layers and well-organized

epithelial cells. On the other hand, although there was no significant difference in the thickness of cytokeratin between the CPB/ EPC and CPB groups, fewer layers in the neoepidermis can be clearly seen (Fig. 5B), revealing the faster re-epithelialization process in the treated wounds of the scaffold/EPC constructs (Fig. 5C, D). It is worth mentioning that even without EPCs, nanofibrous scaffolds alone still have a positive effect on reepithelialization, as shown by shorter wound length and thicker cytokeratin layer in the CPB and CP groups compared to control. The on-site transplantation of EPCs into the wound sites may stimulate the homing and differentiation of epithelial cells [43], resulting in the higher cytokeratin expression and faster re-epithelialization and further facilitate wound healing. 3.5. Enhanced cell proliferative activities and collagen deposition The above results revealed that CPB/EPC constructs can improve the quality of wound healing by facilitating granulation tissue formation, re-epithelialization, and collagen formation. All these events are directly related to the cell activities involved in wound healing [2]. Here, the expression of Ki67, a biomarker of cell prolif-

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Fig. 6. Masson staining of collagen in wounds treated by CPB/EPC constructs. Rectangles refer to the close-up areas. Original scale bar = 100 mm and close-up scale bar = 10 mm, n = 4 per group.

Fig. 7. Histochemical analysis of collagen I and III expression in wounds treated by CPB/EPC constructs. (A, C) Immunohistochemistry staining images for collagen I and collagen III on days 7 and 14 post-wounding, respectively; (B, D) quantitative analysis of the relative density of collagen I and collagen III on days 7 and 14 after surgery, respectively; Scale bar: 50 mm, # P < 0.05 of CPB/EPC versus CPB groups; n = 4 per group.

erative activities in regenerated tissue [47], was analyzed by immunostaining at day 7 and day 14 (Fig. S3). Positive staining of Ki67 can be seen in all five groups (Fig. S3A), and as expected, there are more numbers of positively stained Ki67 cells in the treated wounds of CPB/EPC constructs than those of any other groups

at both time points. Owing to the high cell proliferative activities, wounds in the CPB/EPC group healed statistically significantly faster and better than the other groups (Fig. S3B). Interestingly, the level of Ki67 moderately declined at day 14 in all groups except control. This is probably because these treated wounds were going

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to exit the proliferation stage and partially enter the remodeling stage of wound healing process, which is consistent with the above H&E and Masson staining results that newly formed granulation tissue became thinner and collagen matrix formed into bundles with an ordered structure. On the other hand, the reduction in proliferative activities at the later stages of healing could prevent the scar hyperplasia and may result in aesthetically satisfactory healing [48]. Collagen deposition and remodeling can increase wound strength and greatly influence the outcomes of healing [2,49]. As the above results showed the promoted cell proliferation in regenerated tissue, whether the collagen deposition can be accelerated was then evaluated by Masson staining. Fig. 6 illustrated the deposition and arrangement of newly formed collagen in the regenerated tissue for all five groups. On day 7, the amount of collagen varied in different groups. Greater amounts of collagen was observed in the treated wounds of CPB and CPB/EPC groups, characterized with strong blue staining of the regenerated tissue, while only smaller amounts of collagen appeared in the CP/EPC group. In contrast, there was no difference between the CP and control groups, which both showed rare collagen fibers in the eschar of the wounds. With the prolongation of healing time, by day 21, collagen deposition increased in all five groups, among which the treated wounds of the CPB/EPC constructs showed the most densely packed collagen fibers that appeared as parallel-like structures. The amount of collagen deposition in the CPB and CP/EPC groups also largely increased with abundant and organized fibers distributed in the wounded area, followed by the CP group with moderate collagen deposition and the control group with still small amount and loosely packed collagen fibers. Masson staining results revealed that CPB/EPC constructs accelerated the deposition of collagen matrix, leading to faster skin regeneration and better healing of full-thickness cutaneous wounds. Because collagen I and III are the dominating components of the ECM in the dermis, their formation plays a vital role in wound healing [50]. Therefore, further immunostaining of collagen I and III in the regenerated tissue was performed, and images are shown in Fig. 7. Results showed that the intensity of collagen I and III expression generally followed a similar pattern as the Masson staining

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results of collagen deposition. Deposition of both collagen I and III increased with the healing time in all five groups; among them, the CPB/EPC constructs displayed a significantly higher intensity of both collagen I (Fig. 7A, B) and III (Fig. 7C, D) deposition at day 7 and day 21 than the CP/EPC and CPB groups, followed by the CP and control groups. Previous studies revealed that normal skin has a higher Col III/Col I ratio when compared with scar tissue, and abundant synthesis of collagen III in the early stage of wound healing is prone to result in scarless healing [51,52]. Together with the HE staining results of skin appendage formation, the appearance of abundant collagen I and III at day 7 and increased expression at day 21 provided a possibility for CPB/EPC constructs to stimulate the regeneration of complete skin that is more similar to the normal one.

3.6. In vivo angiogenic response and mechanism Functional vascularization, which supports the nutrition transportation and oxygen exchange for cells, is crucial for wound repair and regeneration [2,53]. It can be clearly seen that the CPB scaffolds have a potent angiogenesis-promoting effect toward EPCs from the above in vitro results (Fig. 1D, E). However, whether this effect could result in functional blood vessel formation remains unknown. To assess the functionality of newly formed blood vessels in regenerated tissue, laser Doppler and immunostaining were then used to detect the blood flow of wound sites and to evaluate the new vascular networks in wounds, respectively. The CPB/EPC group showed the highest levels of blood flood volume at all four time points among the five groups, and the difference between the CPB/EPC group and other groups was most distinct at the beginning of healing (Fig. 8A). For example, the blood flow of the CPB/EPC group was 869.30 ± 20.20 perfusion units at day 3, which was statistically significantly higher than the CP/EPC (778.67 ± 18. 00), CPB (412.00 ± 11.01), CP (353.30 ± 1.15), and control (340.00 ± 16.09) groups. The level of blood perfusion in all five groups increased from day 3 to day 14 but then decreased at 21 day post-op (Fig. 8B), which is probably because the healing stage partially changed into the remodeling stage after the 3-week healing time (consistent with the Ki67 results).

Fig. 8. Assessment of functional blood vessel formation in wounds treated by CPB/EPC constructs. (A) Laser Doppler scan photographs of the wounds at days 3, 7, 14, and 21 after surgery; (B) Quantification of the blood flow volume by using moorLDI Review V6.1 software; # P < 0.05 of CPB/EPC versus CPB groups; n = 4 per group.

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Fig. 9. Neovascularization evaluation of wounds treated by CPB/EPC constructs. (A) Blood vessels stained with a-SMA (red) and DAPI (blue) and H&E staining in wound bed at day 7 postoperative. Scale bar: 20 mm and 10 mm, respectively; (B) quantitative analysis of vessels pre field at 7 days after surgery corresponding to a-SMA staining; (C) quantitative analysis of vessels pre field at 7 days after surgery in H&E-stained tissues; # P < 0.05 of CPB/EPC versus CPB groups; n = 4 per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. Activation of Hif-1a signaling in wounds treated by CPB/EPC constructs. (A) Fluorescence images of HIF-1a on day 7 in wounds, scale bar: 50 lm; (B) quantification of the cellular immunofluorescence intensity; (C, D, E, F) Western blot analysis and quantification of Hif-1a, VEGF, and SDF-1a proteins; # P < 0.05 of CPB/EPC versus CPB group; n = 4 per group.

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Furthermore, the number of new vascular networks stabilized with smooth muscle cells (SMCs) at day 7 was quantified by staining of alpha-smooth muscle actin (a-SMA) (Fig. 9A). Wounds treated with CPB/EPC constructs showed a stronger positive red staining than other groups with significantly higher vessel number than CP/EPC and CPB constructs in the regenerated tissue (Fig. 9B). Moreover, blood vessels in the CPB/EPC group appeared with a mature structure, with lumen, and in bigger size than those in other groups, which was also confirmed by H&E staining (Fig. 9A, C). The wounds in other groups also showed positive staining of a-SMA, but with less numbers or mature structures of vessels. These results supported the laser Doppler analysis of blood flow, suggesting that CPB/EPC constructs successfully induced angiogenesis in the wound site. Moreover, together with the above data of wound closure rates, wound length, granulation tissue thickness, collagen I and III expression, and blood flow, the CPB/EPC group showed statistically significantly better healing than the CP/EPC and CPB groups. Therefore, it can be concluded that the local delivery of EPCs with CPB could exert a synergistic effect of scaffolds and EPCs, which together promote wound healing. To explore the possible mechanism of the activated angiogenesis in wound tissue treated by CPB/EPC constructs, we detected the level of Hif-1, which is a heterodimeric transcription factor complex consisting of a and b subunits, can regulate cytokines such

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as VEGF and SDF-1a, and plays a vital role in angiogenesis and wound healing [54,55]. In vitro results showed that CPB scaffolds can stimulate the angiogenic ability of EPCs through the activation of Hif-1a/VEGF/SDF-1a cascade signaling (Fig. 2A, B, C). The enhanced angiogenesis of CPB/EPC constructs was also confirmed by the above in vivo study; however, whether Hif-1a regulated the angiogenic process in the treated wounds of CPB/EPC constructs is still unknown. Fig. 10 shows the expression of Hif-1a and protein levels of VEGF and SDF-1a. The staining of Hif-1a appeared very strong in the CPB/EPC groups (Fig. 10A, B), indicating the CPB/EPC constructs activated the expression of Hif-1a in skin wounds. Moderate staining can also be observed in the CP/ EPC and CPB groups, indicating that not only EPCs but also CPB nanofibrous scaffolds can stimulate the expression of Hif-1a, which supported the in vitro results of CPB upregulating the level of Hif-1a in EPCs. In contrast, the presence of Hif-1a was almost negligible in the control and CP scaffold groups, indicating its low expression in these two groups. The protein levels of Hif-1a supported the staining results (Fig. 10C-10–F), displaying the highest band intensity in the CPB/EPC group, followed by the CP/EPC and CPB groups. The expression of VEGF and SDF-1a followed a similar pattern for the expression of Hif-1a, with significantly enhanced levels in the treated wounds of the CPB/EPC constructs. These results indicated that CPB/EPC constructs could enhance

Fig. 11. Schematic of a CPB/EPC construct promoting wound healing. CPB enhances cell proliferation, collagen deposition, differentiation of EPCs through the Hif-1a/VEGF/ SDF-1a pathway, together leading to rapid vascularization and healing in full-thickness wounds.

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blood vessel formation through the Hif-1a/VEGF/SDF-1a pathway [56,57] and further enhance wound healing. On the basis of all the aforementioned results, in this study, we propose one of the possible mechanisms for facilitating wound healing, which is as follows. As shown in Fig. 11, first, the EPCs in the CPB nanofibrous scaffold were activated, and they enhanced the angiogenic ability when cultured in vitro. After transplantation into the wound sites, the angiogenic property of the CPB/EPC constructs was enhanced by local delivery into cells through the activation of the Hif-1a/VEGF/SDF-1a signaling pathway, resulting in efficient delivery of more viable cells into the wound site and rapid neovascularization within the area. With these newly formed formed vessels, cell proliferation, granulation tissue formation, collagen deposition, and remodeling were significantly accelerated, leading to fast re-epithelialization and better healing outcomes with skin appendages appearing in the regenerated tissue. 4. Conclusion In summary, we successfully achieved the efficient local delivery of EPCs with BGN-based bioactive nanofibrous scaffolds to promote wound healing. EPCs cultured on CPB scaffolds showed enhanced adhesion, spreading, and proliferation and expression of angiogenic markers through the activation of the Hif-1a/VEGF/ SDF-1a signaling cascade. In vivo, the CPB/EPC constructs promoted the efficiency and viability of local delivery of EPCs and angiogenesis in the wound site, leading to enhanced cell proliferative activity, granulation tissue formation, and collagen deposition. As a result, better healing outcomes characterized with faster reepithelialization and regeneration of skin appendages were achieved using CPB/EPC constructs, suggesting the highly efficient delivery of EPCs. This study highlights the great potential of using CPB nanofibrous scaffolds as a suitable cell delivery vehicle and CPB/EPC constructs for superior treatment of wound healing. Conflict of interest The authors declare no competing financial interest. Acknowledgements We sincerely appreciate the financial support of the National Natural Science Foundation of China (grant no. U1501245), Zhejiang Provincial Basic Public Welfare Research program (grant no. LGF18H150008), Wenzhou Science & Technology Bureau project (grant nos. Y20160063 and Y20150060), Zhejiang Provincial Medical and Health Technology project (grant no. 2016KYA138), and Zhejiang Undergraduate Talent Project (grant no. 2017R413071). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.actbio.2018.01.019. References [1] A.J. Martí-Carvajal, C. Gluud, S. Nicola, D. Simancas-Racines, L. Reveiz, P. Oliva, J. Cedeño-Taborda, Growth factors for treating diabetic foot ulcers, Cochrane Database Syst. Rev. 10 (2015) CD008548. [2] F.H. Epstein, A.J. Singer, R.A.F. Clark, Cutaneous Wound Healing, New. Engl. J. Med. 341 (10) (1999) 738–746. [3] P. Martin, Wound healing–aiming for perfect skin regeneration, Science 276 (5309) (1997) 75–81. [4] N.B. Menke, K.R. Ward, T.M. Witten, D.G. Bonchev, R.F. Diegelmann, Impaired wound healing, Clin. Dermatol. 25 (1) (2007) 19–25. [5] S. Bennett, G. Griffiths, A. Schor, G. Leese, S. Schor, Growth factors in the treatment of diabetic foot ulcers, British Journal of Surgery 90 (2) (2003) 133– 146.

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