Novel H2S Releasing Nanofibrous Coating for In Vivo Dermal Wound

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Novel HS Releasing Nano-fibrous Coating for In Vivo Dermal Wound Regeneration Jiang Wu, Yi Li, Chaochao He, Jianming Kang, Jingjing Ye, Zecong Xiao, Jingjing Zhu, Anqi Chen, Xiaokun Li, Jian Xiao, Ming Xian, and Qian Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06466 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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ACS Applied Materials & Interfaces

Novel H2S Releasing Nano-fibrous Coating for In Vivo Dermal Wound Regeneration Jiang Wu1,2, Yi Li1, Chaochao He1, Jianming Kang3, Jingjing Ye1, Zecong Xiao1, Jingjing Zhu1, Anqi Chen1, Xiaokun Li1, Jian Xiao1*, Ming Xian3*, Qian Wang2*

1

School of Pharmaceutical Sciences, Key Laboratory of Biotechnology and Pharmaceutical

Engineering, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China

2

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC

29208, USA

3

Department of Chemistry, Washington State University, Pullman, WA 99164, USA

*

Corresponding authors: Jian Xiao, [email protected]; Ming Xian, [email protected]; Qian

Wang, [email protected]

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Abstract Hydrogen sulfide (H2S), together with nitric oxide and carbon monoxide, has been recognized as an important gasotransmitter. It plays an essential physiological role in regulating cyto-protective signal process, and H2S-based therapy is considered to be the next generation of promising therapeutic strategy for many bio-medical applications, such as the treatment of cardiovascular disease. Through electrospinning of polycaprolactone (PCL) containing JK1, a novel pH-controllable hydrogen sulfide (H2S) donor, nanofibers with H2S releasing function, PCL-JK1, are fabricated. This fibrous scaffold showed a pH-dependent H2S releasing behavior, i.e. lower pH induced more and faster H2S releasing. In addition, the H2S releasing of JK1 were prolonged by the fibrous matrix as shown by decreased releasing rates compared to JK1 in solutions. In addition, in vitro studies indicated that PCL-JK1 exhibited excellent cyto-compatibility, similar as PCL fibers. Finally, we investigated PCL-JK1 as a wound dressing toward a cutaneous wound model in vivo and found that PCL-JK1 could significantly enhance the wound regeneration compared with PCL scaffold, likely due to the release of H2S, which results in a broad range of physiological protective functions toward wound.

Keywords: hydrogen sulfide, electrospinning, fibrous materials, polycaprolactone, controlled release, wound healing

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1. Introduction Hydrogen sulfide (H2S), as an important gasotransmitter, has recently been recognized as a signaling molecule, which plays an important role in inflammation, reperfusion injury, and circulatory shock of cardiovascular and nervous systems.1 H2S could rapidly travel through the cell membranes and exert its biological effects, resulting in multiple cytoprotective responses through varied physiological functions,2-3 such as anti-inflammatory through driving macrophage differentiation into anti-inflammatory M2-type macrophage, relaxing vascular, stimulating angiogenesis, and attenuating oxidative-stress-related tissue injury as a scavenger for oxygen, etc.4 Therefore, a lot of efforts have been made to develop the appropriate H2S releasing agents,5 which are commonly termed as H2S-donors or prodrugs. For example, containing active P-S bonds GYY41376 as a slow releasing and water soluble H2S-donor, has been widely studied for its anti-inflammation,7 ion channel regulation8 as well as cardiovascular protection.9

In our group’s previous work, we have successfully incorporated N-(benzoylthio)benzamide (NSHD1) as a H2S donor into polycaprolactone (PCL) to fabricate fibrous scaffolds, which were capable of releasing H2S in a controllable fashion. In addition, the controlled release of H2S could protect fibroblast cells against H2O2 induced oxidative damage and promote expressions of wound healing related genes in vitro.10 However, the H2S-donor, NSHD1, requires biological thiols such as cysteine and glutathione to trigger the release of H2S.11 Although cysteine is prevail in biological systems, there exists uncertainty of the biological thiols that could result in unpredictable and unstable H2S releasing especially for bio-clinical

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applications. Considering this drawback, we recently developed a new series of H2S donors with pH responsive releasing behaviors named as JKs.12 These novel pH-dependent compounds could control H2S release rates based on a novel pH-dependent intramolecular cyclization reaction. For the acute wound tissue, acidic pH immediately takes place with suppressed bacterial growth, reduced proteolytic activity and enhanced fibroblast growth; then it goes back to normal pH with regeneration of wound.13 Therefore, we envision these acidic pH-promoted H2S donors can be used for the treatment of wound healing,14 through in vivo advantages of H2S, including inhibiting inflammation, reducing oxidative damage while increasing angiogenesis .

Herein, electrospinning biodegradable polycaprolactone (PCL) polymers15-16 were doped with a pH-dependent H2S donor JK1, generating PCL-H2S donor fibers.17-18 The so-called PCL-JK1 fibrous material that exhibiting a pH-dependent H2S releasing profile, are employed as novel wound healing scaffolds to enhance wound regeneration. The wound healing effects of PCL-JK1 were assessed using a full-thickness cutaneous wound model with C57BL/6 mice. We found that the resultant PCL-JK1 significantly enhanced wound healing efficiency by releasing H2S compared with PCL fiber.

2. Material and method

2.1. Electrospinning of pH responsive H2S donor fibers H2S donor JK1 was freshly prepared following the reported protocol before electrospinning 12. Polycaprolactone (PCL) (Average

Mn Ca. 60 kDa,

4


Sigma) was dissolved in

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1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Scientific Matrix) to afford a 6% w/w solution; then different amounts of JK1 solution (200 mM) were added to prepare samples with H2S donors (0%, 1%, 5% and 10% to PCL, w/w).

The random nanofibers were obtained by a

home-made electrospinning system through stationary collector described before with a flow rate at 3 µL/min, voltage supply between 10-11 kV, and humidity below 1% 15.

2.2. Fiber characterization The morphology of PCL and PCL-donor fibrous scaffolds were examined by a scanning electron microscopy (SEM, VEGA3 TESCAN). Fiber samples were dried under nitrogen flow before being coated with gold with a Desk II cold sputter coater (Denton Vacuum, Morristown, NJ) for 60 s. At least three areas were randomly selected to test the uniformity of the fibers. In addition, infrared spectrometry of PCL and different PCL-JK1 (1%, 5%, 10%) were taken using a Spectrum 100 (FT-IR) Spectrometer.

2.3. Profile of hydrogen sulfide release under different pH Measurements of release kinetics of H2S from fibrous scaffold were conducted at each time point. For each experiment, 20 mg fibrous scaffold was immersed in 50 mL PBS under different pH (pH 7.4, pH 6.8 and pH 6.0). Reaction aliquots (0.5 mL) were added to mixture of zinc acetate (50 μL, 1% w/v in H2O) and NaOH (6.25 µL, 1.5M) in 1.5 mL centrifuge tubes at certain time intervals. Then centrifuge at 20500 rcf for 1 h, followed by removing the supernatant using a pipette. FeCl3 (100 μL, 30 mM in 1.2 M HCl), and N, N-dimethyl-p-phenylenediamine sulfate (100 µL, 20 mM in 7.2 M HCl) was added to

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centrifuge tubes. At last, solutions are transferred to 96 microplate followed by addition of 1 mL water, and absorbance (670 nm) were taken after 20 mins.

2.4. Cell lines and cell cultures The mouse fibroblast cell line NIT 3T3 was purchased from American Type Culture collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, #D6046, Sigma-Aldrich) supplemented with 10% heat inactivated fetal bovine serum (Hyclone, Thermo Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco BRL, Invitrogen Corp., Carlsbad, CA, USA). Cells were cultured in a 5% CO2 humidified incubator at 37 oC. Sterilized PCL and PCL-Donor fibers were soaked in media for 30 min before cell seeding. Cells were then trypsinized and seeded on PCL or PCL-JK1 nanofibers with cell density of 5×103 cells per cm2.

2.5. Cell viability assay NIH 3T3 was trypsinized and washed by PBS (3×) and resuspended in 1 mL PBS supplemented with Cell TrackerTM deep red dye (2 µM). Cells were stained for 30 min at 37 o

C and washed by PBS (3×) and resuspended in 2 mL DMEM culture medium (pH 7.4 and

pH 6.0 adjusted by HCl). Cells were then counted and seeded in 6-well plate with PCL or PCL-JK1 fibrous scaffold (10% w/w JK1 to PCL) under pH 6.0 and pH 7.4, respectively. Cell density was 40,000 cells for individual well. All cells were incubated under a humidified atmosphere of 5% CO2. At each time points (12 h, 24 h, 54 h, 72 h), cells were directly observed under the fluorescence microscopy (Olympus IX81, Olympus America Inc.).

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2.6. Animal model for wound healing Male C57BL/6 mice weighing 20 g were provided by the Laboratory Animals Center of Wenzhou Medical University, and treated strictly in accordance with international ethical guidelines and the National Institutes of Health Guide concerning the Care and Use of Laboratory Animals. The mouse was anesthetized with 4% chloral hydrate and the skin was cleaned with shaving machine and depilatory creams. Silicone rings with an internal diameter of 8 mm and thickness of 0.5 mm was stitched on the skin. Two full-thickness wounds per mice were created on their mid-back with 6 mm diameter puncher (Acuderm® inc., Ft Lauderdale, FL, USA). Photographs were taken of each wound. PCL fiber and PCL-Donor (10% w/w JK1 to PCL) with diameter of 7 mm were deposited in wound area. Wound were covered with 3M Tegaderm Film (3M Health Care, Germany) and medical bandages. After surgery, photos collection were in days 7, 10, 14, 17, 20 and analysis used Image-Pro plus. The ratio of wound healing was calculated using the equation 1:

(1) where C% is the wound healing closure ratio, C0 is the original wound area, and Cf is the open area on point day. 2.7. Histological staining Skin histological analysis was performed according to previously paper on day 7 and 20 after surgery.19 Briefly, the wound area were picked after anesthesia and euthanized. The skin tissues were fixed in 4% paraformaldehyde at 4 °C overnight then embedded in paraffin, followed by cutting in 5 µm sections with a microtomes (LEICA RM2235, Germany) and put 7



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in 65 °C oven 4 h.

2.8. Hematoxylin-eosin staining The sections were put on the xylene 20 min, 100% alcohol 5 min, 95% alcohol and 80% alcohol 2 min, distilled water 5 min, then hematoxylin (Beyotime Institute of Biotechnology, China) stained nuclear 5 min, PBS washed 3 min to remove excess hematoxylin, eosin (Beyotime Institute of Biotechnology, China) staining extracellular matrix 2 min. After that, the sections washed with distilled water for 5 min, followed by 80% alcohol 2 min, 95% alcohol 2 min, 100% alcohol 5 min, xylene 10 min and covered the tissue use neutral resin. Photographs were taken with Nikon microscope (Nikon, Tokyo, Japan).

2.9. Masson’s trichrome staining The sections were stained using Masson’s trichrome staining kit (Beyotime) following the protocols. In brief, the hydration step was as before described, after 5 min washed with distilled water, stained nuclear with A1:A2 (1:1) 5 min, thoroughly rinsed with water and used acid alcohol differentiation 3 s, then Ponceau acid fuchsin solution stained fibrous tissue 5 min, 2% acetic acid solution soaked 1min and differentiation with phosphomolybdic acid solution 1 min, direct use of aniline blue stained 80 s without washing, sections were mounted dehydration by 80% alcohol 3 s, 95% alcohol 1 min, 100% alcohol 5 min, xylene 10 min and covered the tissue used neutral resin. Photographs were taken with Nikon microscope (Nikon, Tokyo, Japan).

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2.10. Immuno-histochemical staining of cytokeratin Briefly, after dewaxed and hydrated of the tissue sections, 3% hydrogen peroxide (15 min) was used to block the endogenous peroxidase. Primary antibodies for cytokeratin (ab9377, Abcam) were diluted in phosphate-buffered saline (1:200) containing 1% bovine serum albumin (BSA) overnight at 4°C. Biotinylated secondary antibodies were diluted with phosphate-buffered saline (1:1000) and incubated for 60 min in 37°C. DAB kit (ZSGB-BIO, Beijing, China) was used for 8 s to 3 min for all samples as previous reported 20. 2.11. Statistical analysis The statistical analysis was followed the reported methods 19.

3. Results and discussion

Scheme 1. Schematic diagram illustrating the fabrication of PCL-JK1 nanofiber. This novel pH-dependent H2S releasing fibrous scaffold was fabricated through electrospinning. The

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pH-dependent H2S donor JK1 was homogeneously doped in PCL fiber matrix.

3.1 Synthesis and characterization of H2S releasing nanofibers Biodegradable polycaprolactone (PCL) has being frequently used as cellular scaffolds for cell culturing and specific devices in various biomedical applications.21-22 Herein, the PCL and PCL-JK1 fibrous scaffold were fabricated through an electrospinning approach as shown in Scheme 1. The structure of JK1 and the activated H2S-releasing mechanism are shown in Fig. 1A, and this novel pH responsive H2S donor JK1 was incorporated into PCL solution before the electrospinning process. As shown in Fig. 1B, the mixed JK1/PCL solution generated homogeneous nanofibers with smooth and uniform morphology and diameter around 300 nm, which is similar as the fibers generated from pure PCL solution. In addition, we used the FTIR spectrum to further confirm the successful loading of JK1 into PCL-JK1 fibrous scaffold (Fig. 1C). PCL fiber showed peaks at 2950, 2850 and 1720 cm-1 due to the stretching vibration of –C=O bonds. While the PCL-JK1 afforded additional peaks at 3320, 1607 and 720 cm-1, which can be attributed to amide N-H stretch, amide N-H bending and aromatic C-H bending from JK1, respectively. These data confirmed the JK1 donor was incorporated into the nanofibers successfully.

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Figure 1. (A) Proposed mechanism of pH-dependent H2S release from JK1.12 (B) SEM images of electrospun PCL and PCL-JK1 fibers (from top to bottom). Scale bars represent 5 µm. (C) FTIR spectra of PCL fiber, JK1 only, and PCL-JK1 fibers with different amount of JK1 (1%, 5% and 10%). Dash lines show the peaks at 3320 cm-1, 1607 cm-1 and 720 cm-1, attributed to amide N-H stretch, bending and aromatic C-H bending of JK1, respectively.

3.2 pH-controllable hydrogen sulfide (H2S) release profile Since H2S has now been recognized as a potent cytoprotective gasotransmitter, fabrication of bio-compatible scaffolds which can release H2S in a controlled manner could be a promising therapeutic strategy in biomedical applications.23-25 To determine the controlled H2S release profile from PCL-JK1 fibrous scaffold (with 10% JK1) under different pHs (i.e. pH 6.0, 6.8 and 7.4), a modified Methylene Blue method was performed with JK1 in solution as the

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control. As shown in Fig. 2, similar as JK1, PCL-JK1 showed pH-dependent H2S-releasing profile, i. e. lower pH led to higher and faster release of H2S with earlier peaking time (pH 6.0 < pH 6.8 < pH 7.4), as well as higher H2S peak concentration (pH 6.0 > pH 6.8 > pH 7.4). However, notable slower H2S release was observed for PCL-JK1 nanofibers, especially at the early stage in the process (Fig. 2). For instance, under pH 6.0 (Fig. 2B), PCL-JK1 did not produce H2S peak within 120 min. In contrast, JK1 immediately reached peak concentration within 10 min, suggesting extended H2S releasing profiles compared with donor only. These results indicated that PCL-JK1 nanofiber could release H2S in response to solution pH and prolong the H2S release from the donor component.

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Figure 2. H2S release kinetics of donor JK1 versus PCL-JK1 fiber (with 10% JK1) under different pH conditions. (A, C, E) Longer term releasing profiles (0 - 4400 min) under pH 6.0, 6.8 and 7.4 (from top to bottom). (B, D, F) Short term releasing profile (0 - 120 min) under pH from 6.0, 6.8 and 7.4.

3.3 Cyto-compatibility of PCL-JK1 nanofibers Before we applied PCL-JK1 to the in vivo wound model, in vitro investigation of its cyto-compatibility versus PCL was carried out using NIH 3T3 fibroblast cells because 13



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fibroblasts play an important role in wound regeneration process. Since pH value could affect JK1’s H2S release behavior, both pH 7.4 and pH 6.0 were chosen to compare the toxicity of PCL-JK1 upon culturing 12 h, 24 h, 48 h and 72 h in comparison to PCL. We observed no difference in cell viability between PCL and PCL-JK1 at both pH 6.0 and pH 7.4 upon culturing for 72 h (Fig. 3). This data demonstrated that JK1 doped PCL fibers were non-toxic to fibroblast cells, which is essential for the wound healing process. Therefore, we assumed that PCL-JK1 was able to maintain its capacity to support fibroblast cell proliferation in vivo.

Figure 3. NIH 3T3 cell viability tests when cells were cultured for 12 to 72 hours on PCL and PCL-JK1 fibers at pH 7.4 (A) and pH 6.0 (B), showing no significant difference in cell viability.

Furthermore, we used WSP-5 (a fluorescent H2S probe) to monitor the in vitro H2S releasing with fibers.10 As expected, cells cultured on PCL-JK1 nanofibers showed much higher fluorescent signals compared to cells cultured on PCL only (Fig. S1), indicating the release of H2S from PCL-JK1.

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3.4 In vivo wound healing In vivo experiments were then carried out to evaluate the actual wound healing efficacy of this novel H2S releasing PCL nanofiber. It was reported that H2S is a potential gasotransmitter upon wound regeneration because it could promote endothelial cell migration, micro vessel tube formation26 as well as angiogenesis through vascular endothelial growth factor receptor 2 (VEGF-2) pathway.27 Serious damage of skin integrity would cause severe inflammation, losing skin appendages such as vascular and hair follicles, and following by prolonged healing process.28 Thus in this work, full-thickness removal skin caused cutaneous wounds in C57BL/6 mice were created to study the wound healing capability of H2S releasing fibers.

The wound healing progress was analyzed at different time points during 20 days upon treatment. Fig. 4A shows the sequential macroscopic images of full-thickness models treated with PCL and PCL-JK1 scaffolds (with 10% of JK1) for 7, 10, 14, 17 and 20 days. It can be seen that wounds were gradually regenerated from the edge of wound. Compared to PCL treated wounds, PCL-JK1 treated wounds enhanced wound closure at each time point, suggesting the positive function of the PCL-JK1 dressing likely due to the H2S releasing. Quantitatively, Fig. 4B calculated the wound closure rates for both PCL and PCL-JK1 treated wounds. Consistent with visual macroscopic images of wound in Fig. 3A, the healing rate of PCL-JK1 treated group exhibited significantly higher than that of PCL at all time points studied (day 7, 10, 14 and 17). Especially at day 20, the final closure rate for PCL-JK1 treated group was 20% faster than PCL treated wounds, with wound closure rate of 78.7 ± 8.8 % in contrast to 64.8 ± 6.8 %. Both the macroscopic observation and quantified wound

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closure rate revealed that the healing of the wound was significantly improved by treatment with H2S releasing fibers of PCL-JK1 compared to PCL.

Figure 4. Wound closure upon treating with PCL and PCL-JK1 fibrous scaffolds. (A) Macroscopic observation of wounds covered with PCL and PCL-Donor fibers treating on day 0, 7, 10, 14, 17 and 20. The unit of the rulers under the pictures is mm. (B) Wound closure rates for PCL and PCL-JK1 treated wounds. Statistical differences were performed using ANOVA. ** P < 0.01, * P < 0.05, compared to control group, n > 6.

Wounds regeneration comprise granulation tissue formation and re-epithelialization.29 Representative

H&E-stained

histological

formed

granulation

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tissue

images

and

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immunohistochemical staining of cytokeratin images used to evaluate the wound healing progress are shown in Fig. 5. On day 7, PCL group showed very thin granulation tissue layer (Fig. 5A1) and translucent lighter cytokeratin positive cells (Fig. 5A2) and large length of unhealed wound left. While for PCL-JK1 treated group, thicker tissue formation (Fig. 5B1) and deeper cytokeratin positive cells (Fig. 5B2) appeared in the injured wound area with relatively smaller length of unhealed wound, implying PCL-JK1 enhanced quicker wound healing than PCL scaffolds corresponding to migration phase of healing process. On day 20, PCL treated wound still remained larger wound area with thin granulation formation (Fig. 5C1) and insufficient developed epithelialization (Fig. 5C2). While for PCL-JK1 treated group, the newly regenerated dermis and the formed tissues are connected tightly (Fig. 5D1) and filled with sufficient appendant such as hair follicles under fully healed epithelialization layer (Fig. 5D2). And the newly regenerated tissues was very similar to normal skin with fully developed granulation and re-epithelialization. This further indicated that PCL-JK1 matrix is able to facilitate faster and more efficient wound regeneration than PCL fibers towards full-thickness wounds. Furthermore, quantified granulation formation (Fig. 5E) and re-epithelization (Fig. 5F) for PCL and PCL-JK1 were quantitatively analyzed on day 7 and day 20. Again, PCL-JK1 exhibited significantly accelerating healing effects than PCL due to the release of H2S from PCL-JK1 nanofibers.

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Figure 5. Histological analyses of whole wound site upon day 7 and day 20. (A1, B1, C1, D1) HE-staining (Hematoxylin and Eosin-stained) and (A2, B2, C2, D2). Immunohistochemical staining with cytokeratin. The figures share the same scale bar of 500 µm. (E) Quantified granulation formation calculated from HE staining on PCL versus PCL-JK1 on day 7 and day 20. (F) Quantified re-epithelization calculated from cytokeratin staining on PCL versus PCL-JK1 on day 7 and day 20. Statistical differences were performed using ANOVA. ** P < 0.01, * P < 0.05, compared to PCL group, n > 3.

Masson’s trichrome staining with collagen elements in blue, cellular components keratin and muscle fibers in pink, revealed much clearer matricial collagen deposition upon wound 18


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regeneration.30 Fig. 6 depicts the collagen deposition in regenerated skin on indicated time intervals. On day 7, little collagen fibers were shown in PCL (Fig. 6A) treated group either on the edge of wound (magnified i and ii under each post-wound) nor the wound bed (magnified ii under post-wound). However, for PCL-JK1 (Fig. 6B) treated group, much higher collagen deposition was revealed compared to PCL treated group. And some collagen bundles are beginning to appear especially upon wound edge (magnified images of i and iii). On day 20, larger number of collagen bundles and more regular deposition of thicker collagen was presented by PCL-JK1 treated group than PCL group, further indicating the enhanced wound healing capability of PCL-JK1 fibrous scaffold. In addition, quantified evaluation of collagen deposition upon post wound on day 7 and day 20 were shown in Fig. 6E and Fig. 6F, respectively. Deposition of collagen of PCL-JK1 treated group is significantly higher than PCL group on both day 7 (44.5 ± 19.8% versus 5.5 ± 0.6%) and day 20 (62.5 ± 5.6% versus 35.5 ± 5.1%), indicating obvious enhanced neo-tissue formation by doping JK1 to PCL nanofibers.

Furthermore, to evaluate the neovascularization (provide oxygen and nutrients to sustain cell metabolism with wound growing) of wounds, the newly formed vessels were characterized by CD31 (Cluster of Differentiation 31 of vascular endothelial cells marker) staining

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on

day 20 (Fig. S2). Higher immunofluorescence of CD 31 expressions toward wound upon PCL-JK1 coating than that of PCL further demonstrated more and mature newly formed vascular toward wound due to the positive effect of H2S from PCL-JK1. At last, immunohistochemical staining of H2S-generating enzymes (Cystathionine-γ-lyase, CSE)

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was performed to detect the specific role of H2S as a gasotransmitter from PCL-JK1 toward wound. Exogenous administration of H2S would enhance proangiogenic of wound by regulating the expressions of CSE, leading to improved wound regeneration.31 As shown in Fig. S3, compared with PCL fiber, there existed a significant increase of CSE expressions toward wound on day 7 and day 20, suggesting the specific releasing of H2S from PCL-JK1 contributing to the effective wound healing process.

Taken together, compared with PCL fiber, PCL-JK1 was demonstrated to exhibit significantly improved wound recovery efficiency on granulation tissue formation along with wound re-epithelialization and collagen deposition, as well as neovascularization toward wound due to its release of H2S.

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Figure 6. The histological Masson trichrome staining (MTS) revealed collagen deposition upon wound site treated with PCL and PCL-JK1. (A) Wound treated with PCL on day 7. (B) Wound treated with PCL-JK1 on day 7. (C) Wound treated with PCL on day 20. (D) Wound treated with PCL-JK1 on day 20. The figure A-D share the same scale bar of 500 µm. The magnified images of squares i, ii, and iii in A-D are shown under each image with the same scale bar of 100 µm. (E) Quantified expression of collagen density on day 7 for PCL and PCL-Donor. (F) Quantified expression of collagen density on day 20 for PCL and PCL-Donor. Statistical differences were performed using ANOVA. ***P < 0.001, compared to PCL group, n = 6.

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4. Conclusion The PCL fibrous matrix that doped with a novel pH-controllable H2S releasing donor JK1 was used as a wound healing scaffold to accelerate wound regeneration by releasing H2S. The obtained PCL-JK1 hybrid nanofibers showed pH regulated H2S releasing behavior as well as comparatively slower releasing rate by contrast to JK1 in solution, and excellent cyto-compatibility in vitro. Further in vivo study showed that this hybrid PCL-JK1 dressing exhibited obvious promoted dermal regeneration compared with PCL fibers being applied into the full-thickness removal wound healing model of C57BL/6 mice. Our data demonstrated that PCL-JK1, as a H2S donor doped matrix, could indeed promote wound healing efficiency through H2S’s unique cyto-protective characteristics in vivo, likely due to special biological effects of H2S such as inhibiting inflammation, reducing oxidative damage and increasing angiogenesis. At the moment, it is still hard to pinpoint how the pH-dependent release of H2S contribute to different stages of the wound healing process. More systematic studies are undergoing to address this important issue in our group. Finally, as many of the effects of GYY4137 (a similar phosphine-sulfide based donor compound) are now being attributed to the phosphine-oxide side product, more control studies will be performed to confirm the physiological rule of H2S with careful designed in vivo experiments.

Acknowledgement J.W. is indebted to the financial support from Wenzhou Science & Technology Bureau of China (Y20140727), Zhejiang Provincial Natural Science Foundation of China (LQ15E030003), and the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (YKFJ001). M.X. acknowledge the financial support

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of NIH (R01HL116571). J.X. acknowledge the financial support of Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. QW acknowledge the support of NSF ECCS-1509076.

Supporting Information The experimental section of detection of H2S release and immunohistochemical staining of CD 31 and CSE; three supporting figures. The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org.

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TOC figure 146x145mm (150 x 150 DPI)


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Novel H2S Releasing Nanofibrous Coating for In Vivo Dermal Wound

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