Histatin1-modified thiolated chitosan hydrogels enhance wound healing by accelerating cell adhesion, migration and angiogenesis

Journal Pre-proof Histatin1-modified thiolated chitosan hydrogels enhance wound healing by accelerating cell adhesion, migration and angiogenesis Zhen Lin (Conceptualization) (Methodology) (Data curation) (Formal analysis) (Funding acquisition) (Software) (Writing - original draft) (Writing - review and editing), Riwang Li (Conceptualization) (Methodology) (Data curation) (Formal analysis) (Software) (Writing - original draft) (Writing - review and editing), Yi Liu (Software) (Visualization) (Investigation) (Formal analysis) (Validation), Yaowu Zhao (Software) (Investigation) (Supervision) (Project administration), Ningjian Ao (Conceptualization) (Supervision) (Validation) (Funding acquisition), Jing Wang (Conceptualization) (Funding acquisition) (Writing - review and editing), Lihua Li (Conceptualization) (Methodology) (Funding acquisition) (Writing review and editing), Gang Wu (Conceptualization) (Resources) (Supervision)

PII:

S0144-8617(19)31378-5

DOI:

https://doi.org/10.1016/j.carbpol.2019.115710

Reference:

CARP 115710

To appear in:

Carbohydrate Polymers

Received Date:

3 March 2019

Revised Date:

25 November 2019

Accepted Date:

5 December 2019

Please cite this article as: Lin Z, Li R, Liu Y, Zhao Y, Ao N, Wang J, Li L, Wu G, Histatin1-modified thiolated chitosan hydrogels enhance wound healing by accelerating cell adhesion, migration and angiogenesis, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115710

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Histatin1-modified thiolated chitosan hydrogels enhance wound healing by accelerating cell adhesion, migration and angiogenesis

Zhen Lin a, #, Riwang Li b, c, #, Yi Liu d, Yaowu Zhao e, Ningjian Ao c, Jing Wang a,*, Lihua Li b, *, Gang Wu f

a

Department of Orthopedics, The First Affiliated Hospital of Jinan University, Guangzhou 510630,

b

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China. Department of Material Science and Engineering, Engineering Research Center of Artificial

Organs and Materials, Jinan University, Guangzhou 510632, P. R. China.

Institute of Biomedical Engineering, College of Life Science and Technology, Jinan University,

Guangzhou 510632, P. R. China. d

Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatological Hospital

School of Stomatology, Jinan University, Guangzhou, 510632, P. R. China

Department of Oral Implantology and Prosthetic Dentistry, Academic Centre for Dentistry

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f

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of Guangzhou Medical University, Guangzhou, China. e

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Amsterdam (ACTA), VU University Amsterdam and University of Amsterdam, MOVE Research

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These authors contributed equally to this work.

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Corresponding authors.

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*

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Institute, 1081 LA Amsterdam, Nord-Holland, The Netherlands.

Graphical Abstract

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Highlights

The hydrogels are thermosensitive with in-situ injection performance.

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The CSSH/Hst1 hydrogel can enhance cell adhesion, migration and angiogenesis.

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The Hst1 peptide loaded hydrogels can be utilized for promoting wound healing.

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Abstract

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It is urgently needed for effective treatments of extensive skin loss, wherein lack of angiogenesis is a major obstacle. In this study, we present a thermosensitive thiolated chitosan (CSSH) hydrogel conjugated with Histatin1 (Hst1) as a wound

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dressing to study its efficacy in enhancing the cell adhesion, spreading, migration, and angiogenesis. The composite hydrogels with gelation time of 5 to 7 min, showed a

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prolonged release of Hst1. Cell culture indicated that the adhesion, spreading, migration and tubule formation of HUVECs were promoted, especially for the Hst1-H group. The in vivo healing evaluation showed that the rate of recovery in Hst1-H group was increased to 84% at day 7, and the CD31 positive cells, vascular endothelial growth factor (VEGF) positive cells and aligned collagen fibers were significantly more than the controlled groups. Therefore, CSSH/Hst1 hydrogel is a promising candidate for wound healing by accelerating cell adhesion, migration and 2

angiogenesis. Keywords Histatin1; Chitosan; Cell adhesion; Migration; Angiogenesis; Wound healing 1. Introduction Wounds are a major health problem throughout the world (X. Li et al., 2017).

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There are 6.5 million patients suffering from wounds in the United States (Xiao et al., 2016). Wound healing is a complex and well-orchestrated procedures which is

disrupted in the healing process of extensive skin defect (Eming, Martin, & TomicCanic, 2014). Wounds are difficult to healing due to the impaired vascular and

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migration function. The healing process can be accelerated by stimulating cell

adhesion, migration and angiogenesis. In addition, a moist condition is beneficial to

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wound healing (Dumville, Stubbs, Keogh, Walker, & Liu, 2015). Thus, it is necessary to develop bioactive materials with the ability to keep moisture condition, to enhance

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cell adhesion, migration and angiogenesis, thus promoting the wound healing process. Compared to skin, oral wounds heal faster and show less scarification and

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inflammation, possibly because of proteins in the gingiva promoting migration and angiogenesis (Kou et al., 2018). Histatins, a human-specific peptide in the gingiva, are thought to accelerate wound healing in vitro (Shah, Ali, Shukla, Jain, & Aakalu,

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2017). Histatin1 (Hst1), one of these histatins, significantly enhances adhesion and spreading of epithelial cells (van Dijk, Ferrando, et al., 2017). Moreover, Hst1

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strengthens epithelial barrier function by enhancing cell to cell adhesion (van Dijk, Nazmi, Bolscher, Veerman, & Stap, 2015). More recently, Hst1 was found to enhance adhesion, migration and angiogenesis of endothelial cells in vitro (Torres et al., 2017). Both migration and angiogenesis are crucial for wound healing, making Hst1 an interesting candidate for wound healing. However, Hst1 peptide is unstable with short half-life in vivo. Moreover, bolus release of Hst1 may weaken the repairing effect. Therefore, the combination of Hst1 and matrix would solve these challenges 3

simultaneously. To enhance wound healing, negative pressure wound therapy is widely applied to accelerate angiogenesis, migration and to maintain moist environment. However, the need for repeated application affects the healing process and leads to prolong hospital stays, drive up costs and create long-term pain for patients (Game et al., 2016). Therefore, a single application simultaneously accelerating cell adhesion, migration and angiogenesis is important to clinical use. The single application not only reduces the pain and discomfort caused by frequent dressing changes but also saves significant

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healthcare resources. Hydrogels, composed of a large amount of water, have been used to facilitate wound healing (L. Zhao et al., 2017). Chitosan, one of the most used biomaterial, have several advantages, such as good biocompatibility, low cost,

antibacterial, degradable, and gelation ability (Chen et al., 2017). However, the pure

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chitosan hydrogels do not have the ability to enhance cell migration and angiogenesis. In addition, chitosan hydrogels show poor cell adhesive properties due to the smooth

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surface. To resolve these challenges, we sought to fabricate a wound-dressing biomaterial that could facilitate wound healing by (i) providing a new matrix for cell

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adhesion, (ii) promoting cell migration, (iii) accelerating angiogenesis. In the present study, Hst1 peptide was conjactated with an in-situ formable

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thermosensitive chitosan hydrogels. Cell proliferation, spreading, migration and angiogenesis on the composite hydrogel were firstly evaluated in vitro. To detect the efficacy on wound healing, the hydrogels were implanted into the full-thickness

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defect wounds in rat. Re-epithelialization was detected by Hematoxylin-eosin (H&E) and Masson trichrome staining. To further evaluate the angiogenesis of wound

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healing, angiogenic markers were assessed by immunohistochemistry. We expect the novel Hst1 composite hydrogels can be utilized for rapid healing of large area wound. 2. Materials and methods 2.1. Materials Chitosan (CS, medium molecular weight, Mw  = 190 – 310 kg/mol, degree of 4

deacetylation = 75 – 85 %), L-cysteine hydrochloride monohydrate (Cys) were obtained from Sigma-Aldrich. Beta-Glycerophosphoric acid disodium salt pentahydrate (β-GP), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), N-Hydroxysuccinimide (NHS) were purchased from Qi Yun Biotechnology Co., Ltd. (China). 4% Paraformaldehyde purchased from Beijing Labgic Technology Co., Ltd. (China). The Rhodamine B modified Hst1 (Rhodamine B-Hst1), the peptides Histatin1 (Hst1: DSPHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN, ≥ 95% pure) were

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manufactured by Shanghai Top-peptide Biotechnology Co., Ltd. 2.2. Preparation of CSSH, CSSH hydrogels and CSSH/Hst1 hydrogels

CSSH (Thiolated Chitosan) was synthesized as description in our earlier

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publications (R. Li, L. Deng, et al., 2017). Briefly, 1% (w/v) chitosan was dissolved in 0.5% (v/v) acetic acid solution, and stirred overnight. Then, 50 mM of EDAC, NHS

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were added, and stirred 15 min, the Cys (the molar ratio of chitosan to Cys is 1:1) was added and the pH was adjusted to 5.0 – 6.0 with 1 M NaOH solution. After incubated

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for 5 hours at room temperature under stirring, the resulting conjugate was dialysed (MWCO 8–14 kDa) in the dark, and finally lyophilized (SCIENTZ-10N freeze dryer).

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The degree of substitution (DS) of CSSH was 6.0 % determined by 1H NMR. The CSSH hydrogels and CSSH/Hst1 hydrogels were formed as following. The weighed CSSH was completely dissolved in pH = 8.0 aqueous solution and chilled, then β-GP

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was added with stirring to achieve a homogenous solution with the pH of about 7 and chilled until use. Hst1 completely dissolved in deionized water with concentrations of

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125 μg/μL and 12.5 μg/μL, and the Hst1 solution was added respectively to the CSSH solution with magnetic stirring of 500 rpm for 60 min, the volume ratio of Hst1 solution to CSSH solution is 1 : 9 (v/v). Finally, the CSSH hydrogels and CSSH/Hst1 hydrogels were formed at 37 °C in water bath, the final concentrations of CSSH was 5 wt%, Hst1 were 12.5 μg/μL and 1.25 μg/μL, and were denoted as CSSH Gel, Hst1-H (High) and Hst1-L (Low), respectively. 5

2.3. Characterization of CSSH, CSSH hydrogels and CSSH/Hst1 hydrogels The freeze-dried sponges of CSSH, CSSH Gel and CSSH/Hst1 hydrogels was characterized by Fourier transform infrared spectroscopy (FT-IR) using an EQUINOX55 spectrometer (Bruker, Germany) equipped with an attenuated total reflectance (ATR) accessory. The gelation time was determined by using the vial inverting method and rheological measurements. The rheological measurements used a Kinexus Pro rheometer with parallel plates (Ø 20 mm, Malvern, UK). The time-sweep of precursor

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solution was carried out at 37 °C, a frequency of 1 Hz, and a strain of 1 % (R. Li, Z. Cai, et al., 2017; Ye et al., 2016).

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2.4. In vitro release of Hst1.

The hydrogels formed by 0.4 mL gel precursor with Rhodamine B and

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Rhodamine B modified Hst1 (Rhodamine B-Hst1) were used to imitate the release profiles of Hst1 from Hst1-loaded hydrogels (Van Tomme, van Nostrum, de Smedt,

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& Hennink, 2006; Zhang, Tao, Li, & Wei, 2011). To measure the in vitro release profile of Rhodamine B and Rhodamine B-Hst1, the hydrogel samples were immersed in 3 mL release medium of pH = 7.4 phosphate buffer, and shaken in the shaker at

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37 °C in water bath at 150 rpm. At specific time intervals, 3 mL release medium was withdrawn and replaced with fresh medium. To calculate the total cumulative amount

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of Rhodamine B and Rhodamine B-Hst1 released from hydrogels, three parallel samples were measured by a UV spectrophotometer at 550 nm. The cumulative

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release (%) of Rhodamine B and Rhodamine B-Hst1were calculated using the equation below: 𝑛

Cumulative release = (3 × ∑ 𝐶𝑖 ) / 𝑀0 × 100 % 𝑖=1

Where M0 is the initial mass of Rhodamine B and Rhodamine B-Hst1 in the samples and Ci is the concentration of Rhodamine B and Rhodamine B-Hst1 released 6

at each sampling time point. 2.5. Cell adhesion and spreading The cell adhesion and spreading of Human Umbilical Vein Endothelial Cells (HUVECs) on the hydrogels surface was evaluated as described previously (van Dijk et al., 2015). 40 μL hydrogels containing different concentration of Hst1 were transferred into 96-well plate. The cell spreading assay was performed as described in the literature. Briefly, 5×103 cells were seeded in per well with medium without FBS.

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After 1 and 3 days, the cells were washed and then fixed in 4% Paraformaldehyde.

Cell number was counted in a microscope field with magnification of 200×. Six fields were randomly chosen to calculate the ability of cell adhesion and spreading of

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hydrogels. 2.6. Cell proliferation

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The proliferation behavior of HUVECs and mouse embryonic fibroblasts cells

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(NIH3T3) were evaluated in different dissolution product of hydrogels. The extracted method has been used in previous studies (Y. Wang et al., 2016; S. Zhao et al., 2015). Briefly, 100 mg lyophilized hydrogels were extracted in 1 mL serum-free Dulbecco's

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modified Eagle's medium (DMEM) (GIBCO, Australia). The extracted liquid was centrifuged after incubating in medium for 24 h at room temperature. Then the

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supernatant was filtered and added to 10% FBS and 1% penicillin/streptomycin (GIBCO, Australia).

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The Hst1 released from the hydrogels was used to evaluate cell proliferation, migration and tube formation. The proliferation was evaluated using Cell Counting Kit-8 (CCK-8) proliferation assays. 5×103 cells were seeded per well and incubated 1, 3, and 7 days. Then 10 μL CCK-8 solution and 90 μL culture medium were added to each well. Aliquots (100 μL) was taken from each well at 37°C for 2h and transferred to another 96-well plate. The absorbance at 450 nm was measured with a spectrophotometric microplate reader (Bio-Rad 680, USA). 7

2.7. HUVEC migration and tube formation Transwell assay was used to evaluate the effect of extracted Hst1 on HUVEC migration (Torres et al., 2017). Briefly, 2×104 cells were seeded in the upper chamber of a 24-well Transwell plate (Corning; pore size = 8 μm). Then 600 μL of medium without FBS containing the extracted liquid from different groups was added to the lower chamber. The upper chamber was removed and erased with a cotton swab. The lower surface of the chamber was fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 10 min. Five randomly selected fields per filter were evaluated.

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Matrigel™ matrix (BD Bioscience, CA) was used to assess the tubule formation activity of Hst1 on HUVECs (Torres et al., 2017). Briefly, 50 μL Matrigel per well

was added to the tubule formation plate (Corning; USA). 8×103 cells were added to

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each well and cells were treated with different extracted solution from each group of

2.8. In vivo wound healing.

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Hst1 hydrogels. The nodes were counted with microscope after 5 h.

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Twelve male Sprague-Dawley (SD) rats weighing 300-350 g were used to evaluate the wound healing characteristics of hydrogels. 3% pentobarbital sodium solution (1 mL/kg) was administered intraperitoneally in order to anesthesia. Firstly,

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dorsal area of the rats was carefully shaved and sterilized. Then, 2 circular wounds was punched with a diameter of 5 mm of each mouse by a biopsy punch. 40 μL

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sterilized CSSH Gel, Hst1-L and Hst1-H hydrogels were implanted into each wound,

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respectively. Wounds covered without any hydrogels were used as the control groups. 2.9. Measurement of wound healing. The wounds were photographed at day 2, 5 and 7 post-surgery. The closed

wound area was measured by two independent researchers using an image analysis software (Image J). A0 was defined as the initial wound area. The reserved wound area (At) at each observation time point was carefully traced along the wound margin and the traced area was then calculated using Image J software. 8

The percentage of closed wound area (% of closed wound area) was determined using the formula: Percent of closed wound area = (A0 − At)/A0 × 100 %. 2.10. Histological, immunohistochemical and immunofluorescence analysis After 7 days of healing, the wound tissues including the entire wound with adjacent normal skin were removed. The samples were fixed in 4% paraformaldehyde and embedded in paraffin. Then, the samples were sliced at a thickness of 5 μm, the

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sections were stained with hematoxylin and eosin (HE) and Masson’s trichrome for histological analysis (Toda et al., 2008). The length of epithelial gap and neo-

epithelium were measured using Image J software. The epithelial gap was defined as the distance between the edges of unhealed epidermis. The length of neo-epithelium

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was simply calculated by length of initial wound minus length of the epithelial gap.

Immunohistochemical staining were used to evaluate the angiogenesis potential.

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For immunohistochemistry staining of CD31 and VEGF, the tissue sections were rehydrated and incubated with antigen retrieval. Then, primary antibody solution

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containing rabbit anti-CD31 (1:100, Servicebio, China) or rabbit anti-VEGF (1:100, Servicebio, China) was used to incubate the samples at room temperature for 2 h.

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Subsequently, secondary antibody was used to stain the sections. The nuclei were stained with hematoxylin. After staining, the sections were analyzed using a light microscope (Leica DM 2500, Germany). To quantitate the number of blood vessels,

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the vessel density was calculated by counting the vessel numbers in a microscope

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field with magnification of 200×. Three fields were randomly chosen in each sample to calculate the angiogenesis of wounds (Y. Wang et al., 2016). 2.11. Statistical Analysis Results were expressed as mean ± SD. All results were statistically analysed by analysis of variance (ANOVA) using GraphPad Prism 5. A value of P < 0.05 was considered statistically significant. 9

3. Results 3.1. Properties of hydrogels A thermosensitive chitosan system by incorporation of β-GP has been reported (Liu et al., 2014). Fig. 1A showed the hydrogels precursors, which are flowing at room temperature. After incubation at 37 °C, the liquid became hydrogels as showed in Fig. 1B. The gelation time of the hydrogels were between 5 min and 7 min by the vial inverting method at 37 °C in vitro. When we were doing animal experiments, we

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recorded the gelation time of the in-situ injectable forming hydrogels in vivo were

about 8 minutes. In addition, we also used rheological experiments to analyze the gel time point. The time-sweep of the Hst1-H precursor solution can be seen from Fig.

1E, the storage modulus (G’) gradually increased and it was observed to intersect loss

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modulus (G’’), revealing the gel begins the gelation process, the gelation time point of

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Hst1-H hydrogel was about 40 s (Fig. 1F). Therefore, we can know that the gel can form quickly in situ, and can be used for the repair of skin defects in vivo.

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We also examined the effect of temperature on the gel. As can be seen that the CSSH hydrogel formed at 37 °C (Fig. 1C), then the hydrogel was placed at -20 °C for a period of time and taken out, and it was observed that the gel softened as the

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temperature dropped (Fig. 1D). Therefore, we can see that the hydrogels are thermosensitive and have in-situ injection forming performance.

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The reaction mechanism of CSSH Gel formation was confirmed by Fourier transform infrared spectroscopy. As shown in Fig. 1G, 2360 cm-1 is the characteristic

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peaks of free thiol groups of CSSH, 1525 cm-1 is the amide bond of CSSH. After gelation, the free thiol groups reacted to form the disulfide bonds, and formed hydrogen bonding between CSSH and β-GP, so the peaks of 2360 cm-1 and 1525 cm-1 decreased. When the Hst1 was added to form the gel, it can be seen that the amino peak of Hst1 appears at 1546 cm-1. This indicated that the Hst1 peptide was successfully compounded in the thiolated chitosan hydrogels gel matrix by the interaction with hydrogen bonds. Therefore, CSSH Gel formed by the formation of 10

disulfide bonds between thiol groups of CSSH, and the hydrogen bonds between

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CSSH and β-GP.

Fig. 1. Sol-gel phase transition photographs of CSSH at room temperature (A), and 37 °C (B). Photos of the CSSH Gel at 37 °C (C), and was placed at -20 °C for a

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period of time and then taken out (D). (E) Storage modulus (G’) and loss modulus

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(G”) analyses of Hst1-H hydrogels during gelation process (37 °C, frequency: 1.0 Hz, strain: 1.0 %), and the corresponding local curve in the initial 600 s (F). (G) FT-IR

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spectra of CSSH, CSSH Gel and Hst1-L hydrogels. 3.2. Releasing behavior of Hst1 composite hydrogels

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Rhodamine B and Rhodamine B-Hst1 were used to simulate the release process of the Hst1 peptide from hydrogels. The standard curve of Rhodamine B: A =

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0.07604 C + 0.00049, R2 = 0.99996. As shown in Fig. 2A, the release amount of Rhodamine B increased with incubating time and was almost completed within 3 days. However, the release of low concentration of Rhodamine B-Hst1 (Rhodamine B-Hst1-L) was about 74%, the high concentration Rhodamine B-Hst1 (Rhodamine BHst1-H) was about 50% after 3 days, and the remaining Hst1 peptides were slowly released over time. The cumulative release rate of Rhodamine B-Hst1 is lower than that of Rhodamine B after 3 days. From the corresponding local release curve of Fig. 11

2B, we could see the release rate was fast in the initial 12 h, then became slow. In addition, the release rate was decreased with the increase of Rhodamine B and Rhodamine B-Hst1 concentration. It can be seen from Fig. 2C that the red color of the Rhodamine B-Hst1 hydrogels slowly faded with the release of the Rhodamine BHst1. Therefore, the release of Hst1 peptide is through the diffusion mechanism or degradation process of the hydrogels, and the residual of the Hst1 will be released

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through the diffusion and the degradation of chitosan in vivo.

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Fig. 2. (A) Cumulative release profile of Rhodamine B and Rhodamine B-Hst1 from hydrogels. (B) The corresponding local release curve of Rhodamine B and Rhodamine B-Hst1 from hydrogels in the initial 12 h. (C) Images of hydrogels before

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and after release of the Rhodamine B-Hst1.

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3.3. Cell adhesion and cell spreading of Hst1 composite hydrogels Cell adhesion and spreading of HUVECs on different hydrogels were evaluated.

Hst1-H hydrogels were found to accelerate cell adhesion and to enhance the percentage of spreading cells (Fig. 3A-E). In Fig. 3B, HUVECs adhesion was improved 1.53-fold and 1.63-fold for Hst1-H hydrogels when compared with CSSH hydrogels at day 1 and day 3 (Fig. 3 B&C). However, Hst1-L hydrogels did not cause 12

any increase or decrease on cell adhesion at any time compared to CSSH hydrogels. Afterwards, we examined whether Hst1 composite hydrogels affect cell spreading. HUVECs were plated on different hydrogels and incubated for 1 and 3 days. Then cells were scored as “round” or “spreading” as described previously (van Dijk et al., 2015). Cell spreading was enhanced approximately 2.29-fold and 1.75-fold for Hst1-H hydrogels when compared with CSSH hydrogels at day 1 and day 3 (Fig. 3D). However, Hst1-L hydrogels had no effect on cell spreading compared to CSSH

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hydrogels.

Fig. 3. (A) HUVECs attachment on the chitosan hydrogels in the presence or absence

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of Hst1 peptide. Quantification of HUVECs cell adhesion to three groups of hydrogels after 1 (B) and 3 (C) days. Quantification of HUVECs cell spreading on

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three groups of hydrogels after 1 (D) and 3 (E) days. Mean ± SD; n = 6. * p<0.05, ** p<0.01, *** p<0.001 compared to CSSH hydrogels, # p<0.05, ## p<0.01, ###

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p<0.001 compared to Hst1-L hydrogels. 3.4. Cell proliferation, migration and angiogenesis of dissolution product of Hst1 composite hydrogels The proliferation behavior of HUVECs and mouse embryonic fibroblasts cells (NIH3T3) in different dissolution product of hydrogels were shown in Fig. 4A (HUVECs) and Fig. 4B (NIH3T3). Results showed that the absorbance of cells 13

significantly increased with the culture time (P < 0.05). Moreover, the proliferation of cells on Hst1-L and Hst1-H hydrogels showed no significant difference with that of CSSH hydrogels at 1, 3, 7 days (P > 0.05). These results indicated that Hst1 composite hydrogels had good cell biocompatibility. As shown in Fig. 4C, the migration and angiogenesis ability of HUVECs were evaluated. The migration rate was significantly enhanced in the dissolution product of Hst1-H hydrogels compared with that of CSSH hydrogels (P < 0.05). Moreover, the migration rate of Hst1-H hydrogels was 2.22-fold and 1.66-fold higher than that of

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CSSH hydrogels and Hst1-L hydrogels, respectively (Fig. 4D). Tubule formation of HUVECs was evaluated to determine the angiogenesis of

Hst1 releasing form different hydrogels. HUVECs incubated with Hst1 from Hst1-H

and Hst1-L hydrogels resulted in tube-like structures, however, HUVECs treated with

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dissolution product of CSSH hydrogels formed incomplete or sparse tubular networks (Fig. 4E). In addition, cells in Hst1-H hydrogels group showed more tube formation

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than Hst1-L and CSSH hydrogels (P < 0.05).

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Fig. 4. HUVECs (A) and NIH3T3 (B) cells were incubated with dissolution product

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releasing from three groups of hydrogels. After 1, 3 and 7 days, the proliferation of HUVECs and NIH3T3 cells was evaluated by CCK-8 assays. (C) The effect of Hst1

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peptide dissolution product releasing from three groups of hydrogels on transwell migration and tubule formation of HUVECs. (D) Quantitation of HUVECs migration using a transwell assay. (E) Quantitation of tubule formation by counting the number of complete capillaries connecting individual points of the polygonal structures. Mean ± SD; n = 6. * p<0.05, ** p<0.01, *** p<0.001 compared to CSSH hydrogels, # p<0.05, ## p<0.01, ### p<0.001 compared to Hst1-L hydrogels. 15

3.5. Hst1 composite hydrogels enhancing wound healing in vivo The effect of the composite hydrogels on wound healing in vivo was evaluated at 0, 2, 5 and 7 days. As shown in Fig. 5A and B, the wound area in all four groups became smaller with increasing time and the wound healing of Hst1-H hydrogels was significantly faster than that of Con, CSSH hydrogels and Hst1-L hydrogels at day 2. The rate of recovery of was enhanced to 84% in Hst1-H hydrogels group, which was higher than those for the CSSH hydrogels (70%) and Hst1-L hydrogels (72%) at day 7.

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The wound healing of different composite hydrogels was further evaluated by

HE and Masson’s trichrome staining. Histological analysis showed Hst1-H hydrogels exhibited the best repair outcome of the wounds among the four treatment groups. As

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shown in Fig. 6A&C, the length of the unhealed epithelium in Hst1-H hydrogels

treated group was 0.34 mm, which was significantly shorter than that of the CSSH

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hydrogels (1.19 mm) and Hst1-L hydrogels (1.09 mm) at day 7 in HE staining. Meantime, the length of neo-epithelium in Hst1-H hydrogels treated group was 4.66

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mm, which was significantly longer than that of the CSSH hydrogels (3.81 mm) and Hst1-L hydrogels (3.91 mm) in Fig. 6D. The same trend was observed in Masson’s trichrome in Fig. 6B&E&F.

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Moreover, as shown in Fig. 7, collagen fibers were in ordered arrangement in the wounds treated with Hst1-H hydrogels, while these were distributed randomly in the

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wounds treated with CSSH hydrogels and Hst1-L hydrogels. These demonstrated that

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the positive effect of the Hst1 on extracellular matrix (ECM) remodeling.

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Fig. 5. (A) Representative images of full-thickness skin wounds on day 0 (D0), day 2

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(D2), day 5 (D5) and day 7 (D7) after treatment with no hydrogels (Con), with peptide-free hydrogels (CSSH hydrogels), with a low dose of Hst1 peptide immobilized to hydrogels (Hst1-L), with a high dose of Hst1 peptide immobilized to hydrogels (Hst1-H). (B) Percent wound closure for the wounds at 7 days post-surgery. Mean ± SD; n = 3. * p < 0.05 compared to Con.

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Fig. 6. Representative images of H&E (A) and Masson's trichrome staining (B) in the wounds treated with no hydrogels, with peptide-free hydrogels (CSSH hydrogels),

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with a low dose of Hst1 peptide immobilized to hydrogels (Hst1-L), with a high dose of Hst1 peptide immobilized to hydrogels (Hst1-H) on day 7. The double-headed

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arrows indicated the length of the unhealed epithelium. (C&D) Quantification of epithelial gap and length of neo-epithelium from H&E staining collected 7 d after treatment. (E&F) Quantification of epithelial gap and length of neo-epithelium from

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Masson's trichrome staining collected 7 d after treatment. Mean ± SD; n = 3. * p < 0.05 , ** p < 0.01 compared to Con. # p < 0.05 compared to CSSH hydrogels. & p <

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0.05 compared to Hst-L hydrogels.

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Fig. 7. Collagen fibers were ordered arrangement in the wounds treated with a Hst1-H

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hydrogels. Representative images of H&E and Masson's trichrome staining. 3.6. Hst1 composite hydrogels promoting angiogenesis in wounds healing

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To detect the effect of Hst1 composite hydrogels on angiogenesis, the angiogenic markers (CD31 and VEGF) were detected in the wound area by

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immunohistochemical staining. There were no statistically significant differences regarding the expression of CD31 among the Con, CSSH hydrogels and Hst1-L hydrogels groups (P > 0.05). However, the CD31 positive cells of the Hst1-H

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hydrogels group were significantly higher than those of Con group (p < 0.05). The

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expression of VEGF showed the similar tendency (Fig. 8C). The VEGF positive cells of the Hst1-H hydrogels group were significantly higher than those of Con and CSSH hydrogels groups (p < 0.05). Results demonstrated that the wounds treated with Hst1H hydrogels exhibited more intensive blood vessels nets compared with that of the Con and CSSH hydrogels at day 7.

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Fig. 8. Effect of Hst1 hydrogels on angiogenesis in the full-thickness wounds.

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Representative images of immunohistochemical staining of CD31 and VEGF (A) at day 7 post-surgery. Arrows indicated CD31-positive or VEGF-positive cells.

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Quantification of number of CD31(B) and VEGF(C) in the wounds treated with no hydrogels, with peptide-free hydrogels (CSSH hydrogels), with a low dose of Hst1 peptide immobilized to hydrogels (Hst1-L), with a high dose of Hst1 peptide

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immobilized to hydrogels (Hst1-H) on day 7. Mean ± SD; n = 3. * p<0.05, ** p<0.01

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compared to Con. # p < 0.05 compared to CSSH hydrogels.

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4. Discussion

In our studies, we combined thermosensitive chitosan hydrogels and Hst1

peptide to form a novel wound dressing. The release of Hst1 increased with time and released more than half of the amount within 3 days, which is consistent with our previous studies (R. Li, L. Deng, et al., 2017). The Hst1 had no effect on cell proliferation but enhanced the adhesion and spreading of HUVECs on hydrogels. In addition, the dissolution product of the Hst1 hydrogels stimulated the migration and 20

tubule formation of HUVECs. Compared with the untreated group and CSSH hydrogels group, Hst1-H hydrogels showed a better capacity to stimulate angiogenesis and to accelerate the healing of the full-thickness skin wounds in rats. Negative pressure wound therapy is commonly applied to accelerate wound healing which is beneficial for adhesion, migration and angiogenesis of skin tissue. However, the shortcomings of this therapy, such as expensive and time-consuming, restrict its applications. Hydrogels have been widespread applied as wound-dressing materials for its good biocompatibility (T. Wang et al., 2017). However, hydrogels

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have been reported to have poor cell affinity. The adhesive RGD peptide was used to modified hydrogel to improve cell adhesion (Kudva, Luyten, & Patterson, 2018). Recently, mussel-inspired Dopamine (DA) have been widely used in surface

modification of biomaterials (Alas, Agarwal, Collard, & Garcia, 2017). Previous

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studies have applied DA to modify hydrogels to enhance adhesive properties, thus accelerating wound healing (Pandey et al., 2018). In our study, Hst1 peptide was

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incorporated into hydrogel to enhance cell affinity. Compared with other adhesive molecules, Hst1 have several prominent advantages. Firstly, Hst1 possessed good

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biocompatibility. Secondly, Hst1 enhance cell attachment without changing the intrinsic physicochemical characteristics of hydrogels. Thirdly, Hst1 enhance cell

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migration and angiogenesis at the same time, which are critical for wound healing. Cell attachment to the implant surface is important for obtaining rapid tissue to implant integration and good aesthetics (van Dijk, Beker, et al., 2017). Conventional

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strategies to improve implant integration are based on implant surface modification (Correia, Gaifem, Oliveira, Silvestre, & Mano, 2017). Recently, modified surface

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with cell adhesion peptides has been studied extensively (van Dijk, Beker, et al., 2017). Previous studies demonstrated that Hst1 significantly enhances spreading of epithelial cells on glass. Moreover, Hst1 promoted cell to cell adhesion at the same time (van Dijk et al., 2015). Both cell-surface attachment and cell-cell adhesion play critical role in implant integration. Here we applied the cell-stimulating properties of Hst1 peptide to enhance cell adhesion and spreading on hydrogels. Results showed Hst1 hydrogels enhanced cell adhesion and spreading of the different cell types on 21

hydrogels, including endothelial and fibroblast cells. The proliferation of cells was evaluated using CCK-8 assay. In our study, the proliferation of cells significantly increased with the culture time. In addition, extracting solution of Hst1-H hydrogels showed negligible effect on cells viability when compared with CSSH hydrogels. These results demonstrated that hydrogels composed of Hst1 had low cytotoxicity, which is important for further clinical translation. It is well known that migration and angiogenesis of endothelial cells are

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important for wound healing (Y. Wang et al., 2016; S. Zhao et al., 2015). Our main hypothesis of the present study is that the Hst1 peptide released from hydrogels are not only enhance cell affinity but also have the activity to stimulate migration and angiogenesis. Our results demonstrated that the dissolution product of the Hst1-H

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hydrogels significantly enhanced migration and tubule formation when compared with CSSH hydrogels. These results are consistent with previous reports for Hst1

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enhancing endothelial cells migration and angiogenesis in vitro (Torres et al., 2017). Re-epithelialization plays an important role in wound healing (Xiao et al., 2016).

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Previous study demonstrated that Hst1 could enhance wound healing in vitro by accelerating cell migration. In our study, a significant improvement in wound closure

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was observed in the defects treated with Hst1-H hydrogels at each time point postsurgery when compared with that of the untreated defects and CSSH hydrogels. By day 7, the regeneration of the epidermis was almost complete in the defects treated

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with the Hst1-H hydrogel. Approximately 84% defect site was covered with healthy epithelial tissue in the defects treated with the Hst1-H hydrogel. The higher

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percentage of epithelialization, as confirmed by the shorter epithelium gap in the H&E and Masson's trichrome staining. Staining also showed more mature granulation tissue in the defects treated with the Hst1-H hydrogels than that in the defects treated with the Hst1-L hydrogels, CSSH hydrogels or in the untreated defects. Accelerating angiogenesis is a dominating aim of therapeutic interventions in wound healing (Dohle et al., 2018; Hu et al., 2018). In our study, immunochemical staining for VEGF and CD31 were used to evaluate the newly-formed vessels. The 22

results of immunohistochemistry staining indicated that treatment of the skin defects with the Hst1-H hydrogels not only enhanced wound healing but also improved the number of newly-formed blood vessels (Fig. 8). Wound healing was a complicated process and usually contributed by wound reepithelization, angiogenesis and wound contraction in mice. In our study, would healing process possibly benefited by wound contraction. However, the skin defect model used in this article was not perfect for evaluating the process of wound contraction. To avoid the effect of wound contraction, the silicone sheet fixed wound

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model will be used in our next study. Local application of growth factors, such as fibroblast growth factor (FGF),

epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) is the

most common procedure to enhance wound healing by accelerating angiogenesis (Qu

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et al., 2018; Xue, Zhao, Lin, & Jackson, 2018). However, skin defect repairing is complicated process which needs synergistic effect of cell adhesion, spreading,

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migration and angiogenesis (Xiao et al., 2016). Therefore, many of these growth factors failed to achieve complete wound healing. Hence, we focused on applying a

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growth factor that would promote the adhesion, spreading, migration and angiogenesis at the same time. In conclusion, the Hst1 peptide promoted cell

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adhesion, spreading, migration and angiogenesis in vitro. In vivo, the Hst1 peptide mixed with hydrogels accelerated wound healing by enhanced re-epithelialization and angiogenesis. The hydrogels composed with Hst1 peptide as a therapeutic candidate

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for promoting wound healing. In our study, the Hst1 hydrogels significantly enhance angiogenesis and migration of cells, which would possibly be beneficial for the

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healing of wound. 5. Conclusions

We designed an in-situ formable CSSH/Hst1 hydrogel to benefit wound healing. The prolonged release of Hst1 peptide was found to increase the HUVECs adhesion, spreading, migration and angiogenesis in vitro. Animal implantation in vivo revealed 23

that the wounds treated with Hst1-H hydrogels exhibited faster wound closure, more angiogenic markers, aligned collagen fibers and more intensive blood vessels nets. Therefore, CSSH/Hst1 hydrogel is a promising candidate for wound healing by accelerating epithelialization and angiogenesis. Conflict of interest

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The authors declare that they have no conflict of interest.

Acknowledgements

This study was financially supported by grants from the science and technology

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project of Guangdong (Grant 2017A020211026), the fundamental research funds for the central universities (Grant 21617312), the medical science and Technology

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Author Contribution

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Research Fund Project of Guangdong (Grant A2018333).

Zhen Lin: Conceptualization, Methodology, Data curation, Formal analysis, Funding

Editing.

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acquisition, Software, Writing-Original draft preparation, Writing-Reviewing and

Riwang Li: Conceptualization, Methodology, Data curation, Formal analysis, Software,

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Writing-Original draft preparation, Writing- Reviewing and Editing. Yi Liu: Software, Visualization, Investigation, Formal analysis, Validation.

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Yaowu Zhao: Software, Investigation, Supervision, Project administration. Ningjian Ao: Conceptualization, Supervision, Validation, Funding acquisition. Jing Wang: Conceptualization, Funding acquisition, Writing-Reviewing and Editing, Lihua Li: Conceptualization, Methodology, Funding acquisition, Writing-Reviewing and Editing. Gang Wu: Conceptualization, Resources, Supervision. 24

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