In-Situ-Generated Vasoactive Intestinal Peptide Loaded Microspheres

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In-situ Generated Vasoactive Intestinal Peptide Loaded Microspheres in Mussel-inspired Polycaprolactone Nanosheets Creating Spatiotemporal Releasing Microenvironment to Promote Wound Healing and Angiogenesis Yuzhen Wang, Zhiqiang Chen, Gaoxing Luo, Weifeng He, Kaige Xu, Rui Xu, Qiang Lei, Jianglin Tan, Jun Wu, and Malcolm M.Q. Xing ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11332 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 26, 2016

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ACS Applied Materials & Interfaces

Fig. 1 A: Schematic of detailed chemical process; B: Schematic of fabrication procedure of the PCL-DA-VIP nanosheets with in-situ generated microspheres. Abbreviations: Polycaprolactone (PCL); dopamine (DA); vasoactive intestinal peptide (VIP); VIP-DA-coated PCL (PCL-DA-VIP). 354x245mm (300 x 300 DPI)

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Fig. 2 Representative scanning electron microscopy (SEM) images, the measurement of the VIP-release behavior in vitro and the FTIR-ATR spectra of PCL, PCL-DA and PCL-DA-VIP. Before the treatment of acetone, SEM images showing morphology of PCL-DA (A) and PCL-DA-VIP nanosheets (E). After the treatment of acetone for 10 min (B and F), 1 h (C and G) and 6 h (D and H), microspheres were in-situ generated with the diameters of (2.4 ± 0.7) µm, (1.1 ± 0.3) µm and (0.5 ± 0.1) µm, respectively. Triangles indicate microspheres. Bar: 10 µm. (I): The released VIP (µg/ml) from day 1 to day 5. The values are the mean ± SD (n = 3 per group). (J): The FTIR-ATR spectra of PCL, PCL-DA and PCL-DA-VIP. 234x191mm (300 x 300 DPI)


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Fig. 3 Representative fluorescent images of seeded cells in PCL-DA and PCL-DA-VIP nanosheets at day 3 post-seeding. Arrows indicated cells. Bar: 100 µm. 196x149mm (300 x 300 DPI)



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Fig. 4 Representative SEM images and three-dimensional reconstructed images of seeded cells in PCL-DA and PCL-DA-VIP nanosheets at day 3 post-seeding. (A and D): Cell morphology on the surface of nanosheets. Bar: 50 µm. (B and E): Pseudo colored SEM images showing microspheres (red). Bar: 20 µm. (C and F): Three-dimensional reconstructed images of seeded cells by laser scanning confocal microscopy (LSCM). Triangles indicate microspheres. Arrows indicated cells. The rectangular insets in (A) and (D) indicate the magnified areas. 625x308mm (300 x 300 DPI)


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Fig. 5 The cell number in vitro detected by CCK-8 proliferation assays (450nm). No significant difference of cell number was observed from day 5 to day 9. The values are the mean ± SD (n = 3 per group). 161x169mm (300 x 300 DPI)



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Fig. 6 Representative macroscopic appearance of the wounds: (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. (E) The measurement of closed wound area from day 1 to day 7 post-surgery. The values are the mean ± SD (n = 4 per group). 179x145mm (300 x 300 DPI)


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Fig. 7 The length of the newly-regenerated epidermis at day 3 and day 7 post-surgery. Representative histological images (H&E staining): (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. The double-headed arrows indicate the length of the newly regenerated epidermis. The rectangular insets indicate the magnified areas. (E): The measurement of the length of the newly regenerated epidermis at day 3 post-surgery. The values are the mean ± SD (n = 4 per group). 358x164mm (300 x 300 DPI)



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Fig. 8 The thickness of the granulation tissue at day 3 and day 7 post-surgery. Representative histological images (Masson trichrome staining): (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. The double-headed arrows indicate the thickness of the granulation tissue. The rectangular insets indicate the magnified areas. (Q) The measurement of the granulation tissue thickness at day 3 and day 7. The values are the mean ± SD (n = 4 per group). 383x164mm (300 x 300 DPI)


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Fig.9 The effect of PCL-DA-VIP nanosheets on cell proliferation in the newly regenerated epidermis and the full-thickness wound tissues, respectively. Representative images of immunohistochemical staining of PCNA at day 3 post-surgery: (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. (E): The detection of PCNA-positive keratinocytes in the newly regenerated epidermis at day 3 post-surgery. The values are the mean ± SD (n = 5 per group). Arrows indicated PCNApositive keratinocytes. (F): The protein levels of PCNA in the full-thickness wound tissues, as determined by Western blotting at day 3 post-surgery. (G): The optical density values of the PCNA bands. The values are the mean ± SD (n = 3 per group). 192x155mm (300 x 300 DPI)



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Fig. 10 The effect of PCL-DA-VIP nanosheets on angiogenesis in the full-thickness wound tissues. (A): The protein levels of CD31 and VEGF in the full-thickness wound tissues, as determined by Western blotting at day 3 post-surgery. The optical density values of the CD31 bands (B) and VEGF bands (C), respectively. The values are the mean ± SD (n = 3 per group). 381x114mm (300 x 300 DPI)


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In-situ Generated Vasoactive Intestinal Peptide Loaded Microspheres in Mussel-inspired Polycaprolactone Nanosheets Creating Spatiotemporal Releasing Microenvironment to Promote Wound Healing and Angiogenesis

Yuzhen Wang 1, 2, Zhiqiang Chen 1, 2, Gaoxing Luo 1, 2, Weifeng He 1, 2, Kaige Xu 3, 4, 5, Rui Xu 1, 2, Qiang Lei 1, 2, Jianglin Tan 1, 2, Jun Wu 1, 2, *, Malcolm Xing3, 4, 5, * 1

Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest

Hospital, the Third Military Medical University, Chongqing 400038, China 2

Chongqing Key Laboratory for Disease Proteomics, Chongqing 400038, China

3

Department of Mechanical Engineering, University of Manitoba, Winnipeg MB, R3T 2N2, Canada.

4

Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg MB, R3T 2N2,

Canada. 5

Children’s Hospital Research Institute of Manitoba, Winnipeg, Canada.

Co-first authors: (Yuzhen Wang and Zhiqiang Chen contributed equally to this work) Yuzhen Wang, E-mail address: [email protected]; Zhiqiang Chen, E-mail address: [email protected]. * Corresponding authors: Jun Wu, Tel: 0086-023-68754173, Fax: 0086-023-65461677. E-mail address: [email protected]; Malcolm

Xing,

Tel:

1-204-474-6301,

Fax:

1-204-275-7507.

E-mail

address:

[email protected].

Abstract:

Vasoactive intestinal peptide (VIP) was reported to promote angiogenesis. Electrospun

nanofibers lead to idea wound dressing substrates. Here we report a convenient and novel 1



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method to produce VIP loaded microspheres in polycaprolactone (PCL) nanofibrous

membrane without complicated processes. We first coated mussel-inspired dopamine (DA) to

nanofibers, then used strong adhesive DA to absorb the functional peptide. PCL membrane

was then immersed into acetone to generate microspheres with VIP loading. We employed

high pressure liquid chromatography to record encapsulation efficiency of (31.8 ± 2.2) % and

loading capacity of (1.71 ± 0.16) %. The release profile of VIP from nanosheets showed a

prolonged release. The results of laser scanning confocal microscope, scanning electron

microscope and cell counting kit-8 proliferation assays showed that cell adhesion and

proliferation were promoted. In order to verify the efficacy on wound healing, in vivo

implantation was applied in the full-thickness defect wounds of BALB/c mice. Results

showed that the wound healing was significantly promoted via favoring the growth of

granulation tissue and angiogenesis. However, we found wound re-epithelialization was not

significantly improved. The resulting VIP-DA-coated PCL (PCL-DA-VIP) nanosheets with

spatiotemporal delivery of VIP could be a potential application in wound treatment and

vascular tissue engineering. Keywords: Mussel inspired dopamine, controlled release of vasoactive intestinal peptide, in-situ generated microspheres, wound healing, angiogenesis Abbreviations: 2


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Polycaprolactone (PCL); dopamine (DA); vasoactive intestinal peptide (VIP); DA-coated PCL (PCL-DA); VIP-coated PCL (PCL-VIP); VIP-DA-coated PCL (PCL-DA-VIP).

1. Introduction Skin prevents microbial invasion and keeps the water-electrolyte balance in human body. Extensive skin loss or injury is life-threatening. Better vascular formation can promote the transportation of nutrients and oxygen resulting in accelerated wound healing [1-4]. Diabetic foot ulcers or wounds with bone or tendon exposure, which are characterized by decreased angiogenesis or restricted formation of granulation tissue, are difficult to heal and bring huge medical burden [5]. Although vascular growth factors could be exogenously added to promote angiogenesis, they would be easily decomposed by many activated proteases in the wound tissue as soon as they were applied. Therefore, incorporating vascular growth factors into a polymer matrix and prolonging the releasing of them has been an effective strategy to enhance its in vivo efficiency. To achieve spatiotemporal delivery of growth factors, pre-made microspheres were reported to be loaded into engineered skin scaffolds [6-9]. However, the microspheres were difficult to properly immobilized and homogenously distributed throughout the inner structures. Electrospun nanofibers have the advantage of tunable pore size and large surface/volume ratio to load more bioactive molecules for controlled release. Polycaprolactone (PCL) offers additional advantages over other materials for electrospun scaffolds, including mechanical strength, biodegradability, biocompatibility and ease of biofunctionalisation [10-12]. However, untreated PCL materials are usually hydrophobic and lack of recognition sites for cells or 3



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peptides resulting in poor adhesion of cells or growth factors [13-15]. Some surface functionalization methods have been reported to graft bioactive functional units or drugs in electrospun membranes, such as physical absorption using heparin [16], layer-by-layer polyelectrolyte multilayer assembly using electrostatic forces [17], chemical immobilization using functional amine or carboxylate groups [18], and so on. However, the complicated chemical reaction and electrospun process were always needed and the limited applicability to substrate materials was inevitable [19]. Dopamine (DA) was reported to provide effective adhesive, anchoring, coating and sealant properties in biomedical applications, which was inspired by the mussels’ tough adhesion [20, 21].

Vasoactive

intestinal

peptide

(VIP),

a

28-amino

acid

peptide

(His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Ly s-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2), was reported to promote angiogenesis [22, 23] and wound healing [24]. In this study, we fabricated mussel-inspired electrospun PCL nanosheets embedded with VIP loaded microspheres as a potential application in wound treatment and vascular tissue engineering. During the fabrication, VIP was absorbed onto PCL nanofibers by the adhesive properties of DA [25-27]. The VIP loaded microspheres were in-situ generated so that they were properly immobilized and were distributed homogenously throughout the inner structures. The adhesive properties of DA and the spatiotemporal delivery of VIP were analyzed using high pressure liquid chromatography (HPLC). To detect the biocompatibility for cell adhesion and proliferation in vitro, laser scanning confocal microscope (LSCM) and scanning electron microscope (SEM) were used to show the adhered cells and Cell Counting Kit-8 (CCK-8) 4


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proliferation assays was used to show cell proliferation rate, respectively. To detect the efficacy on wound healing, the PCL-DA-VIP nanosheets were implanted into the full-thickness defect wounds of BALB/c mice. Wound re-epithelialization and the growth of granulation tissue were detected by Hematoxylin-eosin (H&E) and Masson trichrome staining, respectively. To further detect the mechanism of wound healing, proliferation marker (PCNA) and angiogenic markers (CD31 and VEGF) were detected by immunohistochemistry or Western Blotting (WB). 2. Materials and methods 2.1 Materials and animals PCL (MW=70K), acetone, dimethylformamide (DMF), dichloromethane were also purchased from Sigma-Aldrich (MO, USA). Dopamine hydrochloride was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). VIP was purchased from GL Biochem Co., Ltd (Shanghai, China). BALB/c mice (15 to 25g, male) used in our research were purchased from the Experimental Animal Department of the Third Military Medical University. The animals were individually raised for two weeks before the experiments with free access to water and autoclaved rodent chow under standardized conditions (relative humidity: 50%; circadian rhythm: 12 h; room temperature: 25°C). All animal experiments were permitted by the Institutional Animal Care and Use Committee (IACUC) of the Third Military Medical University. 2.2 Preparation of PCL-DA-VIP nanosheets

5



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Fig. 1 A: Schematic of detailed chemical process; B: Schematic of fabrication procedure of the PCL-DA-VIP nanosheets with in-situ generated microspheres. Abbreviations: Polycaprolactone (PCL); dopamine (DA); vasoactive intestinal peptide (VIP); VIP-DA-coated PCL (PCL-DA-VIP).

The fabrication procedure is shown in Fig. 1. Firstly, the PCL nanosheets were fabricated using an electrospinning process as in our previous report [28]. Briefly, PCL was dissolved in DMF/dichloromethane (1:4, v/v) at a concentration of 10 wt. %. The electrospinning process was then carried out with the following parameters: solution feed rate, 1.2 ml/h; applied voltage, 18 kV; syringe needle gauge, 21g; distance between needle and collector, 15 cm; spinning time 4 h. Secondly, the electrospun PCL nanosheets were immersed into a dopamine solution (2 mg/ml, pH 8.5) at 37°C for 18 h to allow dopamine to adhere onto the surface of PCL nanofibers [25, 29]. Thirdly, the dopamine-coated PCL (PCL-DA) nanosheets were washed in purified water and then immersed into a vasoactive intestinal peptide (VIP) solution (1 mg/ml, 6


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pH 7.5) at 37°C for 12 h to allow VIP to adhere to dopamine. After the immersion, the amount of VIP in the reserved VIP solutions (m1) were detected using high pressure liquid chromatography (HPLC). The DA-VIP-coated PCL (PCL-DA-VIP) nanosheets were then washed in purified water. After the washing, the amount of VIP in the washing solutions (m2) were also detected. Lastly, the PCL-DA-VIP nanosheets were immersed into acetone at room temperature to in situ generate the VIP loaded microspheres for 10 min, 1 h and 6 h, respectively. After the immersion, the amount of VIP in the reserved acetone solution (m3) were detected. Therefore, the amount of loaded VIP (mloaded), the loading capacity (LC) and the encapsulation efficiency (EE) were calculated using the following formulas, respectively [30]: mloaded = m0 – m1 – m2 – m3; LC (%) = mloaded / mpcl × 100%; EE (%) = mloaded / m0 × 100%. Where, m0 is the amount of VIP in the initial solutions (1 mg/ml); m1 is the amount of VIP in the reserved VIP solutions after the immersion; m2 is the amount of VIP in the washing solutions; m3 is the amount of VIP in the reserved acetone solution after the immersion; mpcl is the dry weight of the PCL-DA-VIP nanosheets. Three independent samples were tested (n = 3). The microstructures of the prepared PCL-DA and PCL-DA-VIP nanosheets were observed using a scanning electron microscopy (SEM, Hitachi S-3400, Japan). The nanosheets were dried and sputter-coated by Au for 50 seconds and observed at an accelerating voltage of 10.0 kV under high-vacuum conditions. The diameters of microspheres were measured using IPP 7



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6.0 software by two independent researchers. 2.3 Measurement of the VIP-release behavior in vitro The released profile of VIP was detected using high pressure liquid chromatography (HPLC). To detect whether the microspheres could prolong the releasing of VIP, the acetone-treated PCL-DA-VIP nanosheets (with microspheres) and the untreated PCL-DA-VIP nanosheets (without microspheres) were used to detect the VIP release kinetics. To detect whether DA promoted the coating of VIP, we also detected the VIP release kinetics of the VIP-coated PCL (PCL-VIP) nanosheets, which were fabricated by directly immersing PCL nanosheets into VIP solutions (1 mg/ml, pH 7.5) at 37°C for 12 h without the treatment of DA solutions. Briefly, the prepared nanosheets were incubated in PBS at 37 °C under constant shaking. At appropriate intervals (day 1, 3 and 5), the amount of the released VIP was evaluated using a Waters Alliance 2475-2489 HPLC system fitted with a Waters C18 column (Waters, Bridge, Ireland) at a wavelength of 220 nm. The components were eluted at a flow rate of 0.6 mL/min in gradient mode. Chromatography was carried out at 25 °C with mobile phase A H2O (0.1% TFA) and mobile phase-B CH3CN (0.1% TFA). Empower software was used to analyze the raw data. Three independent samples were tested in each group (n = 3). Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) was used to further describe the characteristic of PCL-DA-VIP nanosheets. A Bruker spectrometer (Alpha) with a diamond single reflection attenuated total reflectance (FTIR-ATR) device was used to acquire the spectra. 2.4 The culture and seeding of fibroblasts 8


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Fibroblasts were isolated from the dermis tissue of BALB/c neonatal mice as reported by Cheng et al [11]. Briefly, skin tissues were harvested and washed with PBS and then immersed in 0.5 mg/ml Dispase II (Sigma, USA) at 4°C for 10 h to remove the epidermis tissue. The reserved dermal tissue was cut into pieces and then immersed in 3 ml 0.25 mg/ml trypsin (Boster, China) at 37°C for digestion for 10 min. The digestion was discontinued by adding 5 ml Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) with 10% fetal bovine serum (Gibco, USA). After centrifugation (1000 rpm, 8 min), the suspension was collected and incubated in DMEM containing 10% fetal bovine serum, streptomycin (100 µg/ml) and penicillin (100 U/ml) at 37°C in a 5% CO2 incubator. The PCL-DA-VIP nanosheets were punched into a 4 mm-diameter disc and immersed in 70% alcohol for 1 h for sterilizing and then washed by sterilized PBS for five times. Then, the PCL-DA-VIP nanosheets were immersed in the cell culture medium for 30 min and then removed into a 96-well plate individually. The 4th-passage fibroblast suspension (6.5 × 104/ml, 50 µl) was seeded on one side of the PCL-DA-VIP nanosheets followed with the incubation for about 20 min for cell adhesion. The nanosheets were then turned over and another 50 µl fibroblast suspension was seeded on another side of the nanosheets. The PCL-DA-VIP nanosheets with seeded cells were then incubated at 37°C in a 5% CO2 incubator. The PCL-DA nanosheets and vaseline gauze were used as the parallel groups. 2.5 Evaluation of the cell proliferation in vitro The cells in vaseline gauze, PCL-DA and PCL-DA-VIP nanosheets at day 3 post-seeding were stained by Rhodamine-conjugated Phalloidin (R415, Molecular Probes) and 4′, 6-diamidino-2-phenylindole (DAPI), which could label F-actin (red) and cell nucleus (blue), 9



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respectively [31] and observed using a fluorescent microscope (Leica, CTR6000, Germany). Three-dimensional reconstructed images of cells in PCL-DA and PCL-DA-VIP nanosheets were acquired using a laser scanning confocal microscope (LSCM, Leica Microsystems, Germany) with Leica Confocal Software. The cells on the surface of PCL-DA and PCL-DA-VIP nanosheets were observed by means of SEM. Briefly, samples were fixed in 0.5% glutaraldehyde for 24 hours at room temperature followed by dehydration in a graded ethanol series and then Au sputter-coated for 60 seconds under high-vacuum conditions at an accelerating voltage of 10.0 kV. Cell number in vaseline gauze, PCL-DA and PCL-DA-VIP nanosheets was detected using Cell Counting Kit-8 (CCK-8) proliferation assays at day 1, 3, 5, 7, 9 and 11 post-seeding [2]. Briefly, samples were removed into a new 96-well plate with 100µl culture medium in each well. After incubation for 2 h, 10µl CCK-8 (Dojindo, Kyushu, Japan) solution was added into each well. After incubation at 37°C for 3 h, the mean optical density value of the culture medium was quantitated at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader (Thermo Varioskan Flash, USA) [32]. 2.6 Animal experiment in vivo For anesthesia, 1% pentobarbital were intraperitoneally injected into the BALB/c mice (0.01 mg/g of body weight). The dorsal surface was carefully shaved, sterilized with 75% alcohol and then punched to full-thickness defect wounds with a diameter of 4 mm. The sterilized vaseline gauze, PCL-DA and PCL-DA-VIP nanosheets were implanted into each wound, respectively. All the wounds were then covered with a biological membrane (Negative pressure wound therapy kit, NPWT-1, China) [33]. Wounds covered with only the 10


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biological membrane were used as the control groups. 2.7 Measurement of wound healing As the wound healing was generally measured by the closed area of wounds [2], we therefore photographed the wounds at day 1, 3, 5 and 7 post-surgery and used IPP 6.0 software to measure the closed wound area by two independent researchers. A standard disc with 4 mm diameter was placed close to the wounds and photographed at the same time to represent the initial wound area (I). The reserved wound area (R) was carefully traced along the wound margin and the number of pixels encompassing the traced area was then calculated using the IPP 6.0 software as described by Jang et al [34]. The percentage of closed wound area (% of closed wound area) was calculated using the following formula [2]: % of closed wound area= (I - R) / I × 100%. I is the pixel number of the initial wound area; R is the pixel number of the reserved wound area. 2.8 Hematoxylin-eosin (H&E) and Masson trichrome staining Hematoxylin-eosin (H&E) staining was used to detect the length of the newly-regenerated epidermis, which was defined as the distance between the leading edges of the newly-regenerated epidermis and the edges of unwounded epidermis [35]. Because some wounds in PCL-DA-VIP group have been completely epithelialized at day 7 post-surgery, the length of the newly-regenerated epidermis was measured only at day 3 post-surgery by H&E staining. Masson trichrome staining was used to detect the thickness of the granulation tissue at day 3 and day 7 post-surgery, respectively. Briefly, mice were sacrificed at day 3 and day 7 post-surgery and the full-thickness wound 11



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tissues were carefully harvested, fixed in paraformaldehyde 4% (v/v), embedded with parafﬁn and sliced at a thickness of 5 µm. The sections were stained with H&E or Masson trichrome staining and then were photographed using an optical microscope (CTR6000, Leica, Germany). The length of the newly-regenerated epidermis and the thickness of the granulation tissue, which were crucial indicators for wound healing, were detected using IPP 6.0 software by two independent researchers. 2.9 Immunohistochemistry To detect the proliferation rate of keratinocytes in the newly-regenerated epidermis, the proliferating cell nuclear antigen (PCNA), which is a widely used cell proliferation marker, was immunohistochemically stained at day 3 post-surgery. Briefly, the sections were dried, deparaffinized, rehydrated and incubated in boiling water bath for 30 min and then incubated in 5% H2O2 for 20 min. The sections were washed in PBS three times, incubated in 10% goat serum (Zhongshan Bio-tech Co., Ltd, China) at 37°C for 30min and then incubated with primary antibody against PCNA at 1:800 dilution (ab15497, Abcam, UK) at 4°C overnight. After that, the sections were washed in PBS five times and incubated with biotinylated goat-anti-rabbit IgG antibody (Zhongshan Bio-tech Co., Ltd, China) at 37°C for 40 min, followed by the incubation with avidin peroxidase reagent (Zhongshan Bio-tech Co., Ltd, China) at 37°C for 30 min. The sections were colored with 3, 3'-diaminobenzidine tetrahydrochloride (DAB) solution and were observed using an optical microscope (CTR6000, Leica, Germany). The PCNA positive keratinocytes in the newly-regenerated epidermis per field were counted by two independent researchers. 12


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2.10 Western Blotting To further quantitatively detect the cell proliferation rate and the vascularization, the proliferation maker (PCNA) and the angiogenic markers [CD31 and vascular endothelial growth factor (VEGF)] in the full-thickness wound tissues were tested by Western Blotting at day 3 post-surgery, respectively. Briefly, mice were sacrificed and the full-thickness wound tissues, including the granulation tissue and the newly-regenerated epidermis, were harvested and put into liquid nitrogen immediately for grinding. Pre-cooled RIPA lysis buffer (P0013B, Beyotime) containing PMSF (ST506, Beyotime) was used for protein extraction and BCA protein test kit (P0010S, Beyotime) was used to measure the protein concentration. Protein samples were then added into 12% SDS-PAGE for gel electrophoresis. The isolated protein was then transferred onto PVDF membrane (Tiangen Biotech) at 90 V for 120 min. After the incubation with 5% BSA for 2 h, the membranes were incubated with antibody against PCNA at 1:500 dilution (ab15497, Abcam, UK), antibody against CD31 at 1:500 dilution (ab28364, Abcam, UK), antibody against VEGF at 1:1000 dilution (ab46154, Abcam, UK) and antibody against Tubulin at 1:2000 dilution (ab125267, Abcam, UK) at 4 °C overnight, followed by the incubation with HRP-labeled goat anti-rabbit antibody (1:2000) (Boster, China) at room temperature for 2 h. The protein on the PVDF membranes were then visualized using enhanced chemiluminescence (Pierce, USA) [36]. 2.11 Statistical analysis Results were analyzed by analysis of variance (ANOVA) using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Values are the mean ± SD. The significance level was established as p < 13



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0.05. 3. Results 3.1 Microspheres were in-situ generated in nanosheets after the treatment of acetone

Fig. 2 Representative scanning electron microscopy (SEM) images, the measurement of the VIP-release behavior in vitro and the FTIR-ATR spectra of PCL, PCL-DA and PCL-DA-VIP. Before the treatment of acetone, SEM images showing morphology of PCL-DA (A) and PCL-DA-VIP nanosheets (E). After the treatment of acetone for 10 min (B and F), 1 h (C and G) and 6 h (D and H), microspheres were in-situ generated with the diameters of (2.4 ± 0.7) µm, (1.1 ± 0.3) µm and (0.5 ± 0.1) µm, respectively. Triangles indicate microspheres. Bar: 10 µm. (I): The released VIP (µg/ml) from day 1 to day 5. The values are the mean ± SD (n = 3 per group). (J): The FTIR-ATR spectra of PCL, PCL-DA and PCL-DA-VIP. 14


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SEM images of PCL-DA and PCL-DA-VIP nanosheets before and after the treatment of acetone were shown in Fig. 2A~H. Before the treatment of acetone, PCL-DA and PCL-DA-VIP nanosheets showed a fibrous structure and the nanofibers were about (0.53 ± 0.15) µm in diameter (Fig. 2A and E). After the treatment of acetone, some microspheres were in-situ generated and embedded between the nanofibers (Fig. 2B~D and F~H). To select microspheres with proper diameters, we prepared a series of nanosheets with different time of acetone treatment. Under the condition of the acetone treatment for 10 min, 1 h and 6 h, the diameters of microspheres were (2.4 ± 0.7) µm, (1.1 ± 0.3) µm and (0.5 ± 0.1) µm, respectively, indicating that the diameters of microspheres decreased with the increasing time of acetone treatment. According to the study design, the in-situ generated microspheres should have similar diameters and be homogenously distributed and the general appearance of nanofibers should be complete after immersed into acetone. The results indicated that the microspheres after 10 min of immersion had the varied diameters [(2.4 ± 0.7) µm] and were not homogenously distributed (Fig. 2B and F). The nanofibers after 6 h of immersion were fractured or fused with each other leading to the destroyed porous structures (Fig. 2D and H). After 1 h of immersion, the microspheres had similar diameters [(1.1 ± 0.3) µm] and were comparatively homogenously distributed and the fibrous structures were reserved (Fig. 2C and G). Therefore, the PCL-DA-VIP nanosheets with 1 h of immersion were used for the following experiments. When the PCL-DA-VIP nanosheets were immersed in acetone, some PCL together with the coated DA and VIP got rid of the intermolecular forces in the nanosheets and then were dissolved in acetone. After the immersion, the part of PCL together with the coated DA and 15



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VIP, which had been separated from the nanosheets but had not yet been completely dissolved in acetone, was precipitated onto the surfaces of the nanosheets. Because of surface tension, this part of the precipitated materials developed into PCL microspheres embedded with DA and VIP. As the process of dissolution started at the interface of nanosheets and acetone, a dynamic layer existed, which contained the separated but not yet dissolved materials. When the acetone immersion just started, the materials in the dynamic layer were comparatively in-situ located. Therefore, the microspheres with shorter time of acetone treatment were with larger diameters. With the increased time of acetone immersion, internal structure of nanosheets was gradually dissolved, and materials in the dynamic layer tends to be evenly distributed. Therefore, the microspheres with longer time of acetone treatment were with smaller diameters. The other possibility may be resulted from that the large microspheres could have a further dissolvable status under acetone to get small microspheres due to the time course. 3.2 The loading capacity (LC) and the encapsulation efficiency (EE) The satisfactory encapsulation efficiency (EE) of (31.8 ± 2.2) % (w/w, n = 3) was achieved. The loading capacity (LC) in our study was (1.71 ± 0.16) % (w/w, n = 3), which seems lower than the report of Patil et al. [30]. In our study, the microspheres were in-situ generated and were integrated into the nanosheets, which was different from traditional methods that the microspheres had to be pre-made and then transferred into the nanosheets. Therefore, the LC in our study means the weight ratio of peptides to the whole nanosheets, instead of the weight ratio of peptides to the microspheres. 16


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3.3 Microspheres prolonged the releasing of VIP The release profile of VIP in vitro was shown in Fig. 2I. The released VIP from the untreated PCL-DA-VIP nanosheets (without microspheres) were significantly more than that in other nanosheets at both day 1 (p < 0.01) and day 3 (p < 0.05). However, a significant decrease of the released VIP from the untreated PCL-DA-VIP nanosheets (without microspheres) was observed from day 1 to day 5. The

released

VIP

from

the

PCL-DA-VIP

nanosheets

(with

microspheres)

increased gradually from day 1 to day 5 and were significantly more than that in other nanosheets at day 5 (p < 0.001), indicating that the VIP loaded microspheres prolonged the releasing of VIP. The released VIP from the PCL-VIP nanosheets (with microspheres), in which the VIP were naturally absorbed onto the surface of PCL without the adhesive function of DA, were significantly less than that in other nanosheets from day 1 to day 5, indicating that VIP was difficult to extensively adhere onto the surface of PCL without the adhesive function of DA. The FTIR-ATR spectra of PCL, PCL-DA and PCL-DA-peptide (VIP) were shown in Fig. 2J. In PCL spectrum, the two strong characteristic absorption peaks at 1729cm-1 and 1164cm-1 are from C=O and C-O-C stretching of PCL backbones. After deposition of polydopamine, there are strong and broad multiple peaks appeared around 3100~3500cm-1 contributed to the absorption of O-H and N-H bonds of dopamine, the peak at 1515cm-1 is from C=N bonds of the heterocyclic ring formed during polymerization of dopamine, indicating successful polymerization and deposition of dopamine on PCL substrate. After loading of VIP peptide, the broad peak at 3100 ~3600cm-1 became stronger, which are from absorption of amines of 17



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VIP. The sharp peak appeared at 1650cm-1 are typical stretching vibration of amide groups (N-C=O, amide band I) of VIP peptide, suggesting the loading of VIP peptides on polydopamine. 3.4 Spatiotemporal delivery of VIP significantly promoted cell proliferation in vitro

Fig. 3 Representative fluorescent images of seeded cells in PCL-DA and PCL-DA-VIP nanosheets at day 3 post-seeding. Arrows indicated cells. Bar: 100 µm.

18


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Fig. 4 Representative SEM images and three-dimensional reconstructed images of seeded cells in PCL-DA and PCL-DA-VIP nanosheets at day 3 post-seeding. (A and D): Cell morphology on the surface of nanosheets. Bar: 50 µm. (B and E): Pseudo colored SEM images showing microspheres (red). Bar: 20 µm. (C and F): Three-dimensional reconstructed images of seeded cells by laser scanning confocal microscopy (LSCM). Triangles indicate microspheres. Arrows indicated cells. The rectangular insets in (A) and (D) indicate the magnified areas.

Fluorescence microscope images showed that cells were observed on vaseline gauze, PCL-DA and PCL-DA-VIP nanosheets (Fig. 3A~I). Cell morphology on the surface of PCL-DA and PCL-DA-VIP nanosheets were shown in SEM images (Fig. 4 A, B, D and E). 3D recontructed images by LSCM showed the cell distribution in PCL-DA and PCL-DA-VIP nanosheets (Fig. 4 C and F).

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Fig. 5 The cell number in vitro detected by CCK-8 proliferation assays (450nm). No significant difference of cell number was observed from day 5 to day 9. The values are the mean ± SD (n = 3 per group).

To better understand the effect of spatiotemporal delivery of VIP on both the adhesion and the proliferation of the seeded fibroblasts in vitro, cell number was measured using the CCK-8 proliferation assays. The results showed that the vaseline gauze group had the largest cell number, the PCL-DA group had the fewest cell number and the PCL-DA-VIP group had the middle cell number at both day 1 and day 3 post-seeding (Fig. 5, day 1, p < 0.001; day 3, p < 0.01). The results indicated that vaseline gauze showed the highest efficiency for cell adhesion probably because of the physically tough adhesion ability of vaseline. Moreover, the PCL-DA-VIP nanosheets were significantly more suitable for cell adhesion than the PCL-DA nanosheets. Interestingly, the cell number in the vaseline gauze group was observed to significantly 20


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decrease from day 1 to day 7 post-seeding and a significant increase of cell number was observed after day 7 post-seeding. The results indicated that although vaseline gauze had the highest efficiency for cell adhesion, the cell proliferation was inhibited at early days post-seeding. Although there were fluctuations of cell proliferation line of PCL-DA-VIP nanosheets, no significant difference of CCK-8 absorbance was observed at day 5, 7 and 9 (p > 0.05), which indicated that cell number in PCL-DA-VIP nanosheets was not found to significantly decrease in statistics during culture time. Besides of the effect of nanosheets, many inevitable factors would also effect in vitro cell proliferation, such as cell distribution, cell immigration and cell contact inhibition effect, et al. Therefore, we focused on the final cell number to determine which nanosheets were more suitable for cell proliferation. The PCL-DA-VIP group had the largest cell number than other groups at day 11 post-seeding (Fig. 5, day 11, p < 0.05) and a comparably larger cell number was observed in PCL-DA-VIP group than that in PCL-DA group from day 1 to day 11 post-seeding, indicating that the PCL-DA-VIP nanosheets were more suitable for both cell adhesion and cell proliferation in vitro than PCL-DA nanosheets. 3.5 Spatiotemporal delivery of VIP significantly promoted wound healing in vivo

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Fig. 6 Representative macroscopic appearance of the wounds: (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. (E) The measurement of closed wound area from day 1 to day 7 post-surgery. The values are the mean ± SD (n = 4 per group).

Wound photographs at the designed time points post-surgery are shown in Fig. 6A~D. Wound healing in the PCL-DA-VIP group was found significantly faster than that of other groups (Fig. 6E, day 7, p < 0.05). At day 7 post-surgery, 96.5% of the wound area in PCL-DA-VIP group had been closed. Meanwhile, the percentage of closed wound area in control group, vaseline gauze group and PCL-DA group were 89.25%, 83.25% and 88.25%, respectively. Wound healing in the vaseline gauze group was found significantly slower than that in other group at day 3 and day 5 post-surgery (Fig. 6E, day 3, p < 0.001; day 5, p < 0.001), which probably due to that vaseline gauze inhibited cell proliferation. To detect the mechanism of the promoted wound healing in PCL-DA-VIP group, the length 22


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of newly-regenerated epidermis and the thickness of granulation tissue, which are important indicators of wound healing, were histologically analyzed.

Fig. 7 The length of the newly-regenerated epidermis at day 3 and day 7 post-surgery. Representative histological images (H&E staining): (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. The double-headed arrows indicate the length of the newly regenerated epidermis. The rectangular insets indicate the magnified areas. (E): The measurement of the length of the newly regenerated epidermis at day 3 post-surgery. The values are the mean ± SD (n = 4 per group).

Wound tissue images by H&E staining were shown in Fig. 7A~D. The length of the newly-regenerated epidermis in both PCL-DA group and PCL-DA-VIP group was found significantly longer than that in control group and vaseline gauze group at day 3 post-surgery (Fig. 7E, PCL-DA bar vs. Control bar, p < 0.0001; PCL-DA bar vs. Vaseline gauze bar, p = 0.001; PCL-DA-VIP bar vs. Control bar, p = 0.001; PCL-DA-VIP bar vs. Vaseline gauze bar, p < 0.0001). However, no significant difference of the length of the newly-regenerated epidermis was observed between the PCL-DA group and PCL-DA-VIP group (Fig. 7E).

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Fig. 8 The thickness of the granulation tissue at day 3 and day 7 post-surgery. Representative histological images (Masson trichrome staining): (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. The double-headed arrows indicate the thickness of the granulation tissue. The rectangular insets indicate the magnified areas. (Q) The measurement of the granulation tissue thickness at day 3 and day 7. The values are the mean ± SD (n = 4 per group).

Wound tissue images by Masson staining were shown in Fig. 8A~D. The thickness of granulation tissue in the PCL-DA-VIP group was significantly larger than that in other groups at both day 3 and day 7 post-surgery (Fig. 8E, day 3, p < 0.01; day 7, p < 0.001). 3.6 Spatiotemporal delivery of VIP significantly promoted the cell proliferation and angiogenesis in vivo To detect the effect of PCL-DA-VIP nanosheets on cell proliferation, PCNA was detected either in the newly-regenerated epidermis by immunohistochemical staining or in the full-thickness wound tissues by Western Blotting.

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Fig.9 The effect of PCL-DA-VIP nanosheets on cell proliferation in the newly regenerated epidermis and the full-thickness wound tissues, respectively. Representative images of immunohistochemical staining of PCNA at day 3 post-surgery: (A) control group, (B) vaseline gauze group, (C) PCL-DA nanosheet group and (D) PCL-DA-VIP nanosheet group. (E): The detection of PCNA-positive keratinocytes in the newly regenerated epidermis at day 3 post-surgery. The values are the mean ± SD (n = 5 per group). Arrows indicated PCNA-positive keratinocytes. (F): The protein levels of PCNA in the full-thickness wound tissues, as determined by Western blotting at day 3 post-surgery. (G): The optical density values of the PCNA bands. The values are the mean ± SD (n = 3 per group).

The images of PCNA immunohistochemical staining in the newly-regenerated epidermis were shown in Fig. 9A~D. The PCNA positive keratinocytes in both the PCL-DA group and the PCL-DA-VIP group were found more than that in the control group (Fig. 9E, PCL-DA bar vs. Control bar, p < 0.0001; PCL-DA-VIP bar vs. Control bar, p = 0.001). However, no 25



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significant difference of cell proliferation in the newly-regenerated epidermis was observed between the PCL-DA group and PCL-DA-VIP group (Fig. 9E). The PCNA proliferation in the full-thickness wound tissues, including the granulation tissue and the newly-regenerated epidermis, was detected by Western Blotting (Fig. 9F). The optical density values of PCNA bands of the PCL-DA-VIP group were significantly higher than those of other groups (Fig. 9G, p < 0.001). Although no significant difference of cell proliferation was detected in the newly-regenerated epidermis, cell proliferation was significantly promoted in the full-thickness wound tissues, indicating that spatiotemporal delivery of VIP significantly promoted the cell proliferation in the granulation tissue. To detect the effect of PCL-DA-VIP nanosheets on angiogenesis, the angiogenic markers (CD31 and VEGF) were detected in the full-thickness wound tissues by Western Blotting.

Fig. 10 The effect of PCL-DA-VIP nanosheets on angiogenesis in the full-thickness wound tissues. (A): The protein levels of CD31 and VEGF in the full-thickness wound tissues, as determined by Western blotting at day 3 post-surgery. The optical density values of the CD31 bands (B) and VEGF bands (C), respectively. The values are the mean ± SD (n = 3 per group).

The CD31 and VEGF proliferation in the full-thickness wound tissues were detected by Western Blotting (Fig. 10A). The optical density values of CD31 bands of the PCL-DA-VIP group were significantly higher than those of other groups (Fig. 10B, p < 0.05). The optical 26


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density values of VEGF bands of the PCL-DA-VIP group were significantly higher than those of the control group and the PCL-DA group (Fig. 10C, PCL-DA-VIP bar vs. Control bar, p = 0.001; PCL-DA-VIP bar vs. PCL-DA bar, p = 0.001). However, no significant difference of VEGF was observed between the vaseline gauze group and PCL-DA-VIP group (Fig. 10C). 4. Discussion Wound healing process involves the balanced and timed activity of epithelial cells, dermal cells and vascular tissue [37]. Better angiogenesis was believed to promote wound healing via favoring the proliferation of epithelial or dermal cells. In contrast to use pre-made exogenous microspheres to prolong the spatiotemporally releasing of VIP, the present strategy of in-situ generating endogenous VIP loaded microspheres offers a ready-to-implant graft, which might serve as a therapeutic prototype. In our studies, VIP was incorporated into the in-situ generated microspheres, so that not only the microspheres were properly immobilized and homogenously distributed but also the prolonged spatiotemporally releasing of VIP was achieved (Fig. 2). Moreover, the adhesive function of DA promoted the adhesion of VIP and made VIP more difficult to diffuse from the microspheres, which improved the stability of VIP and pro-longed the release time. Fig. 2I showed that the released VIP from PCL-VIP nanosheets increased rapidly at day 1 and a sharp decrease was observed after day 3. However, the wound healing process lasted more than 7 days in our study (Fig. 6E) and the quickly released peptides would be easily decomposed by many activated proteases. Therefore, the in vivo functions of VIP could not be maintained without the adhesive property of DA. In our study, VIP was firstly absorbed by amido bonds (Fig. 1A) and then embedded in 27



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microspheres (Fig. 2). The amido bonds could be gradually destroyed during cell culture time. Moreover, the microspheres were also gradually degraded. For example, the initial diameters of microspheres were (1.1 ± 0.3) µm (Fig. 2G). After 3 days of cell culture, the diameters of microspheres were (0.33 ± 0.08) µm (Fig. 4E). Therefore, the release mechanism of VIP was possibly due to both the diffusion of VIP and the disassemble of microspheres. The prolonged spatiotemporal delivery of VIP from the implanted PCL-DA-VIP nanosheets yielded a sustained cell proliferating environment for wound healing. The results showed that PCL-DA-VIP nanosheets significantly promoted wound healing compared with control group and vaseline gauze group (Fig. 6), which probably due to the promoted wound re-epithelization (Fig. 7) and granulation tissue formation (Fig, 8). In addition, the promoted wound re-epithelization and granulation tissue formation would be possibly contributed by the promoted proliferation activities of epithelial or dermal cells (Fig. 9). The histological study showed that no significant difference of the length of the newly-regenerated epidermis between PCL-DA and PCL-DA-VIP nanosheets was observed (Fig. 7E), but the granulation tissue was significantly thicker in PCL-DA-VIP nanosheets than PCL-DA nanosheets (Fig. 8E). Relatively, no significant difference of cell proliferation in the newly-regenerated epidermis between PCL-DA and PCL-DA-VIP nanosheets was observed (Fig. 9E), but cell proliferation in the full-thickness wound tissue was significantly promoted in PCL-DA-VIP nanosheets than PCL-DA nanosheets (Fig. 9G). The above results indicated that VIP possibly benefited the formation of granulation tissue, but was not found to benefit the wound re-epithelization. Furthermore, the promoted angiogenesis by the implantation of PCL-DA-VIP nanosheets 28


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would also contributed to the promoted wound healing. The results showed that the angiogenic markers (CD31 and VEGF) in PCL-DA-VIP group were significantly more than that in PCL-DA group and control group (Fig. 10), indicating that the prepared PCL-DA-VIP nanosheets significantly promoted angiogenesis. Vascular endothelial growth factor (VEGF) is a secretory protein and can be produced in many types of hypoxia cells, which are not receiving enough oxygen. The secreted VEGF is circulating among tissues and then binds to VEGF receptors on endothelial cells, triggering a Tyrosine Kinase Pathway and leading to angiogenesis [38]. CD31 is a structural protein and is usually expressed on the surface of endothelial cells, platelets, monocytes, neutrophils, and some types of T-cells, and makes up a large portion of endothelial cell intercellular junctions [39]. Therefore, CD31 expression could represent the actual quantity of vascular tissue, and VEGF expression could comparatively represent the possible ability to promote vascularization because many other vascular growth factors could also induce angiogenesis. Although no significant difference of VEGF was observed between the vaseline gauze group and PCL-DA-VIP group, the most amount of CD31 was observed in PCL-DA-VIP group (Fig. 10C), which indicated that more vascular tissue was generated in PCL-DA-VIP group than vaseline gauze group. Diabetic foot ulcers often lead to lower limb amputation (minimum of one toe) and remain a major therapeutic and financial problem worldwide mainly because of vascular pathologies [40]. Wounds with bone or tendon exposure are difficult to re-epithelialize because of restricted granulation tissue formation. Negative pressure wound therapy was a widely used treatment to keep moist environment in wound beds and wait for the growth of vascular or 29



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granulation tissue [41]. However, this is time-consuming and faces high risks of deep tissue infection. In our study, the PCL-DA-VIP nanosheets showed to significantly promote angiogenesis and granulation tissue formation, which would possibly be helpful for the healing of diabetic foot ulcers or wounds with bone or tendon exposure. Wound healing was complicated process and usually contributed by both wound re-epithelization and wound contraction in mice [2]. In our study, as wound re-epithelization (Fig. 7E) was not found to be significantly promoted, the promoted would healing (Fig. 6E) would be possibly benefited by the promoted would contraction. However, the animal model used in our study was not perfect for observe the process of wound contraction. In our next study, we will use the silicone sheet fixed wound model [42], which can avoid the effect of wound contraction on healing process. 5. Conclusions The mussel-inspired electrospun PCL nanosheets embedded with in-situ generated VIP loaded microspheres could significantly promote wound healing and angiogenesis and could be potentially applied in wound treatment and vascular tissue engineering. Acknowledgments This research was supported by a grant from the National High Technology Research and Development Program of China (863 program) (No. 2012AA020504) and the National Natural Science Foundation of China (No. 81372082). Malcolm Xing thanks the NSERC Discovery Grant for this research. Compliance with ethics guidelines (conflict of interest) The authors declare that they have no conflict of interest. 30


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The graphic TOC:

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In-Situ-Generated Vasoactive Intestinal Peptide Loaded Microspheres

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