Preparation and bioactivity of acetylated konjac glucomannan fibrous membrane and its application for wound dressing

Journal Pre-proof Preparation and bioactivity of acetylated konjac glucomannan fibrous membrane and its application for wound dressing Chuang Wang, Bing Li, Tao Chen, Naibin Mei, Xiaoying Wang, Shunqing Tang

PII:

S0144-8617(19)31071-9

DOI:

https://doi.org/10.1016/j.carbpol.2019.115404

Reference:

CARP 115404

To appear in:

Carbohydrate Polymers

Received Date:

15 May 2019

Revised Date:

24 July 2019

Accepted Date:

29 September 2019

Please cite this article as: Wang C, Li B, Chen T, Mei N, Wang X, Tang S, Preparation and bioactivity of acetylated konjac glucomannan fibrous membrane and its application for wound dressing, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115404

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Preparation and bioactivity of acetylated konjac glucomannan fibrous membrane and its application for wound dressing Chuang Wang, Bing Li, Tao Chen, Naibin Mei, Xiaoying Wang, Shunqing Tang* Biomedical Engineering Institute, Jinan University, Guangzhou 510632, China *Corresponding author: Shunqing Tang, Biomedical Engineering Institute, Jinan University, Guangzhou 510632, China. E-mail addresses: [email protected]

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Graphical abstract

Highlights

Acetylated KGM fibrous membrane was designed as a novel bioactive wound dressing.

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Cells adhered and proliferated better on AceKGM than on KGM.

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AceKGM could enhance the activity of macrophages to accelerate wound healing.

Abstract

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Biomaterial-host interactions significantly affect tissue repair, which is modulated by macrophages. In this study, a polysaccharide, konjac glucomannan

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(KGM), was acetylated with different degrees of substitution (DS), and the acetylated KGM (AceKGM)-based fibrous membrane was designed to modulate the activity of macrophages for accelerating wound healing. AceKGM was biocompatible and easily dissolved in organic solvents. The adhesion force between Raw264.7 cells and the AceKGM substrate was quantitatively detected by atomic force microscopy (AFM). The enzyme-linked immunosorbent assay (ELISA) results showed that the AceKGM fibrous membrane enhanced macrophage expression of anti-inflammatory and 1

pro-regenerative cytokines, and the DS of AceKGM significantly affected membrane bioactivity. The full-thickness mouse skin wound repair experiments indicated that the AceKGM-containing fibrous membranes significantly accelerated wound healing by promoting re-epithelialization, tissue remodeling, and collagen deposition. In summary, AceKGM-based fibrous membranes have potential as bioactive scaffolds for wound regeneration.

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Keywords: Konjac glucomannan, Macrophage, Bioactive fibrous membrane, Growth

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factors, Wound healing

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1. Introduction In recent decades, natural polysaccharides with unique and desirable bioactivities have been frequently used for the design of biomaterials that could promote cell migration, proliferation and the secretion of growth factors. For instance, chitosan was applied for bone and skin repair (Raftery et al., 2016; Wang et al., 2013; Yang et al., 2018) due to its ability to serve as an osteogenic and antibacterial agent. Analogously, β-glucan and acemannan with remarkable immunocompetence were frequently used for skin regeneration (Dianna & Elizabeth, 1995; Lee et al., 2003; Xing et al., 2015).

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KGM, which is composed of D-mannose and D-glucose in a molar ratio of 1.6:1 with β-1,4 linkages, has unique bioactivities and is expected to be used for wound

healing (Sohn & Seo, 2001). KGM is able to stimulate macrophages secreting anti-inflammatory and pro-angiogenic/pro-mitogenic factors via specific affinity

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(Feng et al., 2017; Gan et al., 2018; Gazi & Martinez-Pomares, 2009). KGM has

excellent hydrophilic and gelation capacity and has been widely manufactured into

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films, hydrogels, and control release carriers in food, biomedical and environmental sciences (Alvarez-Manceñido, Landin & Martínez-Pacheco, 2008; And & Nishinari,

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2004; Huang, Chu, Huang, Wu & Tsai, 2015; Nie et al., 2011). Nevertheless, processing KGM into a scaffold is difficult, and KGM has poor stability in the

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physiological environment due to the strong hydrogen bonding between many hydroxyl groups, which limits its application in wound repair. Hence, many reports have recently focused on the modification of KGM (Zhu,

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2018). For instance, Bo et al. (Bo, Muschin, Kanamoto, Nakashima & Yoshida, 2013) reported that sulfated konjac glucomannan was preminently soluble in water and had

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excellent anti-HIV and anticoagulant activities. The carboxymethylation of KGM could increase its hydrophobicity and stability in the physiological environment. Enomoto (Enomoto, 2013) found that the mechanical and thermal properties of KGM acetate could be controlled by tuning the degree of substitution (DS) of the acetyl groups. Acetylation, a simple pattern of intracellular processing, endows biomacromolecules with new bioactivities, and acetyl groups have a significant effect on the bioactivities and other physical properties of polysaccharides. Previous studies 3

showed that acetylation and deacetylation clearly have a significant effect on the solubility, gelation behavior and tensile property of KGM (And & Nishinari, 2004; Gao & Nishinari, 2004; Huang, Takahashi, Kobayashi, Kawase & Nishinari, 2002). Analogously, chitosan, which is derived from the deacetylation of chitin, exhibits biological activities such as wound healing and antimicrobial activity (Muzzarelli, 2009). Acemannan, a polysaccharide extracted from aloe vera gel, could promote wound healing by inducing cell proliferation and upregulation of type I collagen synthesis and vascular endothelial growth factor (VEGF) expression. However, the

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solubility of acemannan decreases in water, and deacetylation reduces the ability of acemannan to stimulate cells expressing VEGF and type I collagen, indicating that the

number of acetyl groups on acemannan has a significant effect on its bioactivity

(Chokboribal, Tachaboonyakiat, Sangvanich, Ruangpornvisuti, Jettanacheawchankit

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& Thunyakitpisal, 2015).

Various biomaterials loaded with growth factors (GFs) have been designed for

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skin (Kanitkar et al., 2016), vasculature and bone tissue regeneration (Kempen et al., 2009); however, the degradation, degeneration, and dosage of exogenous GFs in vivo

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have been major obstacles in clinical application. GFs and cytokines secreted by macrophages play key roles in regulating inflammation, the formation of granulation

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tissue, re-epithelialization, matrix formation and remodeling during wound repair (Barrientos, Stojadinovic, Golinko, Brem & Tomic-Canic, 2008). However, the GFs that are produced by macrophages are massive, multi-type and dynamic in the process

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of tissue repair (Koh & DiPietro, 2011). This sophisticated biological process is hardly mimicked by loading one or several GFs into a biomaterial and delivering it to

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the lesion site. One of the alternative options is to design functional biomaterials stimulating macrophages expressing GFs for tissue repair. In this study, a series of acetylated KGM with different DS were synthesized to

improve the processability and bioactivities of KGM. The fibrous membranes of acetylated KGM (AceKGM) were fabricated by electrospinning. The structures and properties of electrospun AceKGM membranes were characterized. The specific interaction of mannose receptors (MR) on macrophages with various KGM substrates 4

was detected by atomic force microscopy (AFM) at the single-cell level. The AceKGM

membranes

that

stimulated

macrophages

expressing

GFs

were

characterized by enzyme-linked immunosorbent assay (ELISA). The AceKGM-based membranes for wound healing were assessed with a murine full-thickness skin wound model. 2. Materials and methods 2.1. Materials KGM (Mn=6.9×105 Da) was purchased from Chengdu Root Industry Co., Ltd.

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(Chengdu, China). Type I collagen (3×105 Da) was purchased from Mingrang Biotech Co., Ltd (Chengdu, China). Trifluoroacetic anhydride (TFAA), 1,8-diazabicyclo

(5,4,0)-7-undecene (DBU) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) were

purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).

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Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were

purchased from Gibco (Atlanta, GA). The Cell Counting Kit-8 (CCK-8) was

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purchased from Beyotime Biotech (Jiangsu, China). The other chemicals were of analytical grade and were used without treatment. The AFM was a Bioscope Catalyst

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system (Bruker, German). The PNP-TR-TL-SPL AFM probes were purchased from Nano Sensors (Switzerland). The SNL AFM probe was purchased from Bruker

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Corporation (Germany).

2.2. Synthesis of AceKGM

AceKGM was prepared according to a modified preparation procedure from a

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previous study (Enomoto, 2013). First, KGM was purified by being dissolved in deionized water, precipitated by ethanol, treated with Sevag's solution to precipitate

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the proteins and freeze-dried in vacuum. The purified KGM (0.5 g) was added to a premixed solution of TFAA (20 mL) and acetic acid (20 mL) with magnetic stirring at 50 °C for 20 min and then stirred at 50 °C for 1 h under nitrogen gas to yield a homogeneous solution. After being cooled to room temperature, the reaction solution was poured into precooling ethanol (1.0 L) to obtain the precipitate. The precipitate was filtered, washed with ethanol, and dissolved in chloroform, and the insoluble substances were filtered. The products in the chloroform solution were re-precipitated 5

in ethanol, washed with ethanol three times, and dried at 50 °C in vacuum to obtain KGM triacetate. The DS of the obtained AceKGM (abbr AceKGM-3.0) was 3.0 according to the acid-base titration calculations (Koroskenyi & McCarthy, 2001). AceKGM with different DS was prepared by partial deacetylation of AceKGM-3.0. The procedures were as follows: AceKGM-3.0 (0.9 g) was dissolved in dimethylformamide (DMF) (64 mL) under stirring at 40 °C, and then 16 mL methanol and 0.6 mL or 0.3 mL DBU was added to react for 2 h. The reaction solution was poured into 1.0 L precooling ethanol to obtain the precipitate. The precipitate was

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filtered, washed with ethanol, and dried in vacuum to obtain AceKGM with DS =1.0 (AceKGM-1.0) or 1.7 (AceKGM-1.7).

2.3. Fabrication of electrospun AceKGM and AceKGM-1.0/type I collagen fibrous membranes

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AceKGM (2% w/v) was dissolved in HFIP for electrospinning. The solution was

loaded into a 20 mL syringe fitted with a blunted 21G nozzle. The solution feed was

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driven by a syringe pump at a flow rate of 1 mL/h. The distance and DC voltage between the nozzle and the collector (aluminum foil) were 15 cm and 25 kV,

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respectively. All electrospinning experiments were performed at 20 °C and 50% relative humidity. The harvested fibrous membranes were dried overnight in vacuum

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at 50 °C to remove the residue solvents. AceKGM-1.0 and type I collagen with the same weight were mixed in HFIP, and AceKGM-1.0/type I collagen with a weight ratio of 5:5 (abbr A/C55) and type I collagen fibrous membranes were obtained by the

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above electrospinning process.

2.4. Characterization of the electrospun AceKGM fibrous membranes

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2.4.1. Fourier transforms infrared spectroscopy (FT-IR) The KGM or AceKGM samples were analyzed with FT-IR (Bruker Equinox 55,

USA) spectroscopy using potassium bromide pellets. All spectra were obtained with an accumulation of 20 scans in the wavenumber range of 4000–500 cm−1 at a resolution of 4 cm−1. 2.4.2. Scanning electron microscopy (SEM) The morphology of the fibrous membranes was characterized by SEM 6

(LEO1530 VP, Philips, Amsterdam, Netherlands) at an accelerating voltage of 5 kV. Before observation, all the samples were coated with gold using a sputter coater (Leica EM SCD005, Germany). The diameters of the fibers were manually measured from the SEM images by ImageJ (National Institutes of Health, USA). The diameter data were evaluated from at least 50 fibers. 2.4.3. Mechanical properties The tensile strength of the fibrous membranes was characterized by applying a tensile test (AG-I, Shimadzu, Japan). Briefly, all the samples were cut into a

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rectangular shape with dimensions of 10 mm × 50 mm, and the thickness of the sample was detected by a micrometer to be approximately 200 μm. The loading speed

was 10 mm/min along the length direction. The mean ± SD (n = 5) values of the elastic modulus, tensile strength, and elongation at break were extracted from the

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stress-strain curves.

2.4.4. Membrane hydrophilicity and surface energy

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The surface hydrophilicity and surface energy (SE) of the various membranes were characterized by measuring the static contact angles (CA) using a drop shape

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analyzer (KRÜSS, Germany) at room temperature. Three independent measurements were performed for each membrane. The CA and SE were measured using the system

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software, and the average value was reported with its standard deviation. 2.5 In vitro cell experiments

2.5.1 Cell culture and macrophage polarization

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NIH-3T3 and Raw264.7 cells were expanded in complete medium (DMEM, Gibco) supplemented with 10% FBS and 1% streptomycin/penicillin at 37 °C in a

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humidified atmosphere of 5% CO2/95% air. The cells were passaged when they were approximately 80% confluent. The Raw264.7 polarization protocols were as follows: M0 phenotype Raw264.7

cells were cultured in DMEM supplemented with 10% FBS. The M0 phenotype Raw264.7 cells was induced to M1 cells by the presence of 1 μg/mL lipopolysaccharide (LPS) in the complete medium, and was induced to the M2 cells by the presence of 20 ng/mL IL-4 in the complete medium (Odegaard et al., 2007). 7

After 24 h of polarization, the original culture medium was aspirated from all the wells and rinsed twice with PBS and DMEM to remove the polarization medium. 2.5.2 Cell proliferation and morphology All the fibrous membranes were cut into circular disks with a diameter of 15 mm and were fixed on the bottoms of 24-well cell culture plates by Teflon rings. These samples were sterilized by Co60 with 5 kGy radiation. Then, all of the samples were soaked in cell culture media for 30 min, and the NIH-3T3 and Raw264.7 cells were seeded at 5×103 cells per well. Cell proliferation was measured on days 2, 4, and 6

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(n=3/group/time point) by the CCK-8 assay. On day 2, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min, rinsed thrice with PBS, then dehydrated in gradient concentrations of ethanol (50, 70, 80, 90, and 100% for 10 min each), freeze-dried, coated with gold sputter and observed under SEM.

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2.5.3 Cell adherence rate

A suspension of NIH-3T3 and Raw264.7 cells with various phenotypes (M0, M1,

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and M2) was dispersed onto the fibrous membranes in 24-well cell culture plates (2×105 cells per well). The cell adherence rate was measured by the CCK-8 assay

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after incubating for 7 h (n=3/group).

2.5.4 Single-cell force spectroscopy (SCFS)

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The PNP-TR-TL-SPL AFM probe was chemically functionalized according to a previous report for attaching a single suspended cell (Wang, Xie, Huang, Liang & Zhou, 2015). After chemical modification, the probe was stored in PBS at 4 °C and

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used in 1 week. The probe parameters are as follows: nominal spring constant of 0.32 N/m, resonance frequency of 67 kHz, cantilever length of 100 μm, and cantilever

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width of 2 μm × 13.5 μm.

The adhesive forces between the various cells (NIH-3T3 cells and M0, M1, and

M2 phenotype Raw264.7 cells) and substrates were detected by the following protocols. First, the cells were collected, centrifuged and resuspended in PBS; the resuspended cells were injected into Petri dish with PBS. Second, with the aid of an inverted microscope, a modified tip-less cantilever was pressed against a single free cell. The cell was attached to the cantilever by performing a force curve at a contact 8

force of 5 nN, a surface delay of 10 s and an approach and retract rate of 500 nm/s. After the cell attached to the modified cantilever, the cantilever was withdrawn and stayed in PBS for at least 10 min for its surface to firmly adhere to the cell. Finally, force-distance (F-D) curves were recorded using the cell-attached cantilever in ScanAsyst mode with a contact force of 1 nN and a surface delay of 20 s. The approach and retract rate was set at 500 nm/s. After the probe retracted, the cell recovered for approximately 30 s in each F-D curve cycle. Twenty F-D curves were recorded per cell, and 3-5 cells were used for the adhesive force measurements.

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NanoScope analysis software 8.14 was used for processing the F-D curves. 2.6. ELISA

A suspension (1×105 cells per well) of Raw264.7 cells with various phenotypes (M0, M1, and M2) was dispersed onto the AceKGM membranes with different DS in

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24-well cell culture plates. After the cells were incubated for 48 h, the supernatant of

the culture media was collected and centrifuged, and the concentrations of

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pro-inflammatory factors (IL-6 and TNF-α) and anti-inflammatory factors (IL-10 and TGF-β1) were quantified with corresponding ELISA kits with the aid of the

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manufacturer’s instructions.

2.7. Skin wound healing experiment

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The animal experiment was approved by the Animal Care and Experiment Committee of Jinan University. Male Kunming mice (6-8 weeks) were weighed (~20 g) and randomly divided into four groups: untreated group (control), AceKGM-1.0

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group, A/C5:5 group and type I collagen fibrous membrane group (n=5). The mice were anesthetized with sodium pentobarbital by intraperitoneal injection, and their

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dorsal hair was shaved. A full-thickness wound with a diameter of 15 mm was created on the mice dorsal skin, and the wound was sterilized with iodine. A piece of sterilized fibrous membrane 15 mm in diameter was dressed onto the wound. On days 0, 3, 7, 14, and 21, digital photographs of the wounds were taken. 2.8. Histopathological examination On days 3, 7, 14 and 21, the mice were euthanized by cervical dislocation, and the wound samples were harvested and fixed in 4% paraformaldehyde for 24 h, then 9

embedded in paraffin and sectioned perpendicularly to the wound surface in 5 µm consecutive sections using a microtome. The sections were stained with hematoxylin-eosin (H&E) and Sirius Red and imaged with a microscope. 2.11 Statistical analysis All data are shown as the mean ± standard deviation of three independent measurements unless otherwise specified. The data were analyzed with the statistical program Origin 8.5. Statistical analyses were carried out by Student’s t-test between two groups or using one-way analysis of variance (ANOVA) followed by

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Bonferroni’s test among groups. A p-value of less than 0.05 was considered significant. 3. Results 3.1 Synthesis of AceKGM

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KGM with multiple hydroxyl groups is soluble in water but insoluble in organic

solvents. To improve its stability in physiological environments and solubility in

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organic solvents for processing, KGM was modified by acetylation. In the present study, a series of KGM acetate samples with different DS were obtained. The

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structure of AceKGM was confirmed by FT-IR spectroscopy. As shown in Figure 1, the O-H stretching vibration absorption band at 3445 cm-1 decreased, and the C=O

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band at 1750 cm-1 was enhanced, which indicated that the hydroxyl groups on the KGM chain were replaced by carbonyl groups. In addition, the absorption strength of the carbonyl groups increased with increasing DS; in contrast, the absorption strength

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of the hydroxyl groups decreased.

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3.2 Characterizations of AceKGM membranes

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Figure 1. FT-IR spectra of KGM and AceKGM with different DS.

In the process of acetylation, DS had a significant effect on the solubility of

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KGM in water and organic solvents. Either AceKGM-1.0 or AceKGM-1.7 could be soluble in polar organic solvents, such as DMSO, HFIP, and DMF. AceKGM-3.0

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could be soluble in acetone, dichloromethane, and chloroform. AceKGM fibrous membranes with different DS were electrospun with HFIP as a solvent. As shown in Figure 2-A, the AceKGM electrospun membranes exhibited porous structures without

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any beads and branches. The average diameters of the AceKGM fibers with DS of 1.0, 1.7 and 3.0 were 169.8±38.8 nm, 200.1±38.6 nm, and 224.7±71.0 nm, respectively

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(Figure 2-B). Figure 2-C shows the representative stress-strain curves of AceKGM with different DS. The average tensile strengths of AceKGM-1.0, 1.7 and 3.0 fibrous

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membranes were 7.92±0.43 MPa, 3.84±0.36 MPa, and 1.79±0.13 MPa, respectively. As shown in Table 1, the tensile strength and elongation at break values decreased with increasing DS.

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Figure 2. Characterization of KGM and AceKGM membranes. (A, B) Representative SEM

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images and diameter distribution of AceKGM-1.0 (A1, B1), AceKGM-1.7 (A2, B2) and

AceKGM-3.0 (A3, B3). (C) Representative stress-strain curves of AceKGM fibrous membranes with different DS.

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Table 1. Mechanical properties of AceKGM fibrous membranes (n=5). Tensile

Tensile

Elongation

strength/(MPa)

modulus/(MPa)

break/(%)

7.92±0.43

1.7

3.84±0.36

3.0

1.79±0.13

224.85±26.67

6.31±0.64

162.53±14.58

3.16±0.48

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1.0

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DS of AceKGM

79.86±8.66

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2.36±0.34

In this study, the CA values and SE of KGM and AceKGM membranes were

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measured using water and diiodomethane as the probe liquids. The SE of the various KGM membranes and the polar and disperse parts of SE were calculated by the

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Owens-Wendt method (Shimizu & Demarquette, 2000). The results are presented in Table 2. After acetylation, the water CA of the AceKGM membranes obviously

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increased to 74.7±4.1°, 93.2±4.7° and 117.8±4.3°. The SE of the AceKGM membranes decreased with increasing DS. Analogously, the values of the polar component 𝛾𝑠𝑝 remarkably decreased to 9.6, 2.5 and 0.4 mN/m. Table 2. SE components of KGM and AceKGM membranes Sample

KGM

Contact angle (deg)

Surface energy components (mN/m)

Water

Diiodomethane

γs

56.1±3.1

40.8±2.5

48.8±0.8 12

𝛾𝑠𝑑 29.7±0.7

𝑝

𝛾𝑠

19.1±0.3

AceKGM-1.0

74.7±4.1

53.9±2.9

35.8±0.6

26.2±0.4

9.6±0.2

AceKGM-1.7

93.2±4.7

63.2±3.5

26.9±0.3

24.4±0.3

2.5±0.2

AceKGM-3.0

117.8±4.3

69.9±3.4

10.8±0.2

10.4±0.1

0.4±0.1

Here, γs represents the total surface energy of the various KGM membranes, and 𝛾𝑠𝑑 and 𝑝

𝛾𝑠 represent their disperse and polar parts, respectively.

3.3 Cell adhesion, proliferation and morphology in vitro The cytocompatibility of the AceKGM fibrous membranes was evaluated by the adhesion and proliferation of NIH-3T3 and Raw264.7 cells. As shown in Figure 3-A,

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there was no significant difference in the cell adherence rate among the NIH-3T3 cells and M0 and M1 phenotype Raw264.7 cells on the same substrate after incubating for

7 h. However, the M2 phenotype Raw264.7 cells had a higher adherence rate than that of the others. On the other hand, the adherence rate of the cells on AceKGM

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decreased with increasing DS. These results indicated that the AceKGM fibrous membranes had good cytocompatibility and that there was a specific interaction

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between the M2 phenotype Raw264.7 cells and AceKGM.

As shown in Figure 3-B and 3-C, the number of both NIH-3T3 and Raw264.7

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cells increased significantly on the AceKGM fibrous membranes with culture time. Furthermore, cell density on the AceKGM fibrous membranes was higher than that on

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the KGM membranes. The morphology of the NIH-3T3 and Raw264.7 cells on the various membranes is shown in Figure 3-D and 3-E. The NIH-3T3 cells were spindle-like or polygonal in shape. The Raw264.7 cells were round, which indicated

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that the fibrous membranes did not stimulate the Raw264.7 cells to change their

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phenotypes, which was consistent with previous research (Feng et al., 2017).

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Figure 3. Adhesion, proliferation, and morphology of NIH-3T3 and Raw264.7 cells on different KGM or AceKGM membranes. (A) Adherence rate of different cells cultured on KGM or AceKGM membranes for 7 h. (B, C) Proliferation of NIH-3T3 and Raw264.7 cells cultured on KGM or AceKGM membranes for 2, 4 and 6 days. Morphology of NIH-3T3 and Raw264.7 cells cultured on KGM (D1, E1), AceKGM-1.0 (D2, E2), AceKGM-1.7 (D3, E3) and AceKGM-3.0 (D4, E4) membranes for 48 h. Scale bar=50 μm, *p＜0.05, **p＜0.01, ***p＜0.001

3.4 Single-cell force microscopy

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Interaction between the cell and the substrate, such as electrostatic interactions, van der Waals forces and receptor-ligand-specific interactions, play a key role in cell

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adhesion. In the present study, the cell adhesion assay demonstrated a specific interaction between the M2 phenotype Raw264.7 cells and AceKGM, and the cells on

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AceKGM-1.0 had the highest adherence rate. The adhesive force between single cells and AceKGM membranes was quantitatively measured by AFM-SCFS. Figure 4 shows the F-D curves and adhesive forces between the Raw264.7 and NIH-3T3 cells and the AceKGM membranes. The M2 phenotype Raw264.7 cells on the AceKGM-1.0 membranes had the maximum adhesive force (5.77±0.52 nN) (Figure 4-A), and the cells on KGM had the minimum adhesive force (0.67±0.25 nN) (Figure 4-B). There was no significant difference in adhesive force among the NIH-3T3 and 14

M0 and M1 phenotype Raw264.7 cells on the AceKGM-1.0 membranes (Figure 4-C). The adhesive force decreased with increasing DS (Figure 4-D). Nevertheless, the M2 phenotype Raw264.7 cells showed a significantly higher adhesive force than did the other cells. These results suggested that there was a strong cell-substrate-specific

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interaction when M2 Raw264.7 cells were cultured on the AceKGM-1.0 membrane.

Figure 4. F-D curves obtained between M2 phenotype Raw264.7 cells and AceKGM-1.0 (A) or KGM (B). Statistical results of the cell adhesive force between different cells and AceKGM-1.0

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membranes (C) and adhesive force of M2 phenotype Raw264.7 cells on different substrates (D) determined by AFM. The data were obtained with 10–15 cells from 3 separate experiments.

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***p<0.01

3.5 ELISA

A hypothesis of this study was that macrophages could be stimulated by

AceKGM membranes to secrete pro-regenerative cytokines for wound repair. The specific interaction between the M2 phenotype Raw264.7 cells and KGM could be enhanced by acetylation of KGM. An ELISA was performed to detect the levels of inflammatory/pro-regenerative cytokines secreted by Raw264.7 cells with different 15

phenotypes cultured on AceKGM membranes. In Figure 5-A and 5-B, the levels of pro-inflammatory cytokines TNF-α and IL-6 expressed by the M1 phenotype Raw264.7 cells on the substrates were higher than those expressed by the M0 and M2 phenotype cells and were slightly lower on the AceKGM membranes than on the KGM membranes. The M2 phenotype Raw264.7 cells highly expressed anti-inflammatory/pro-regenerative cytokines IL-10 and TGF-β1 on the AceKGM (88 pg/ml) and AceKGM-1.0 (476 pg/ml) membranes, and the levels were significantly higher than those expressed by the control groups (24 and 302 pg/ml, respectively).

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However, the M0 and M1 phenotypes showed fewer factors and no significant

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difference among the groups.

Figure 5. Concentrations of typical pro- and anti-inflammatory cytokines detected in the

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supernatant of different phenotypes of macrophages cultured on various substrates. ***p＜0.01

3.6 In vivo wound healing evaluation To demonstrate the bioactivity of AceKGM in vivo, a dorsal full-thickness skin

defect mouse model was designed. AceKGM-1.0, which was used in the wound healing experiments, has been proven to have good bioactivity and biocompatibility. Figure 6 shows the gross examinations of skin defects at different times. The fibrous membranes were firmly adhered to the defects due to their amphipathic property and 16

porous structures. On the 3rd day, the area of the wound slightly decreased in the AceKGM-1.0 and A/C55 groups. However, there was no significant reduction in the control and type I collagen groups. On the 7th day, all wounds had a scab lesion, and the wound area in the A/C55 group was significantly decreased. On the 14th day, most of the scabs exfoliated except those in the control group. The wound was almost

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closed in the A/C55 groups. All wounds were completely closed on the 21st day.

Figure 6. Assessment of the healing effect of AceKGM in the dorsal full-thickness skin defect mouse model. Representative gross examinations of wounds at 0, 3, 7, 14 and 21 days after treatment of the skin wound in the control, A/C55 and type I collagen groups.

3.7 Histological analysis Figure 7 shows the H&E staining results of the skin wounds at different times. 17

On the 3rd day, a violent inflammation response was observed in the control group, whereas there was only a mild inflammatory response in the AceKGM-1.0 and A/C55 groups. On the 7th day, re-epithelialization proceeded, and most of the wound areas was covered with a continuous epidermis in the AceKGM-1.0 and A/C55 groups, whereas there was an obvious inflammatory response and little epidermis in the control group. On the 14th day, all the wounds were completely covered with epidermis. Neovascularization was observed in the AceKGM-1.0 and A/C55 groups. On the 21st day, substantial epithelialization and neovascularization in the

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regenerative dermis were observed in the A/C55 groups, which were similar to normal

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skin.

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Figure 7. H&E analyses of dorsal skin sections after 3, 7, 14 and 21 days of implantation (magnification: ×100). “S”: scab, “T”: tissue, red arrows: inflammatory cells; blue arrows: epithelialization, scale bar=100 μm.

The synthesized collagen fibers in the wound were analyzed with Sirius Redpicric acid staining. In Figure 8, on the 3rd day, there was only a small amount of regenerative collagen in all the experimental groups. On the 7th day, the amount of type III collagen (green) significantly increased; in contrast, there was only a small 18

amount of type I collagen (red). These results were consistent with those of previous reports (Betz, Nerlich, Wilske, Wiest, Penning & Eiseumenger, 1992; Smith, Holbrook & Madri, 2010). On the 14th day, there was more type I collagen in the AceKGM-1.0, A/C55 and collagen groups than in the control group. On the 21st day, there was no significant difference in the number of collagen fibers among all the groups. As shown in Table S1, the ratio of collagen I/III in the A/C55 groups appeared to be 5.66, which was similar to that of normal skin (3.5) (Chattopadhyay & Raines, 2014). Taken together, these results suggest that AceKGM could promote collagen

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synthesis and neovascularization and accelerate wound healing in vivo.

Figure 8. Sirius Red analyses of dorsal skin sections after 3, 7, 14 and 21 days of implantation (magnification: ×200). Collagen I is shown in red, and collagen III is shown in green.

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4. Discussion Many studies have suggested that chemical modification partially improves the processability of natural polysaccharides and influenced their bioactivity (Niu et al., 2017; Wu et al., 2016). Acetylation is frequently employed to modify polysaccharides. On the one hand, the acetylation of polysaccharides is able to efficiently improve their solubility in organic solvents for easy processing and their stability in a physiological environment. On the other hand, acetylation is a common processing mode in cells, which is able to endow biomacromolecules with new biological activity. The

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acetylation approach is effective in promoting the regulation of antigen presentation and, specifically, inducing macrophages to express abundant cytokines (Broaders, Cohen, Beaudette, Bachelder & Fréchet, 2009). Moreover, acetyl plays a key role in the bioactivity of natural polysaccharides, such as chitosan and acemannan

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(Chokboribal, Tachaboonyakiat, Sangvanich, Ruangpornvisuti, Jettanacheawchankit

& Thunyakitpisal, 2015; Muzzarelli, 2009). To develop a bioactive wound dressing,

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acetylated KGM was successfully synthesized, and its structures and bioactivities were disclosed.

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AceKGM could be processed into a fibrous membrane by electrospinning. As shown in the SEM images, the electrospun fibrous membranes have porous structures that are likely to mimic the physical structure of the extracellular matrix (Guo, Xu,

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Ding, Li, Zhou & Li, 2015). Surface hydrophilicity and SE, which are remarkably impacted by material surface chemical components, are frequently used to evaluate

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material wettability and biocompatibility (Jiang, Zhu, Zhu, Zhu & Xu, 2011). The total SE of KGM membranes decreased with acetylation. Moreover, the polar

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component 𝛾𝑠𝑝 remarkably decreased with increasing DS. This phenomenon was attributed to the substitution of the hydroxy group by acetyl groups, leading to the reduction in the hydrogen interaction between the molecules. The attachment and proliferation performance of NIH-3T3 and Raw264.7 cells on the AceKGM-1.0 and AceKGM-1.7 membranes was better than that on the KGM and AceKGM-3.0 membranes. The results were consistent with those of previous reports in that cells have difficulty in adhering and spreading on excessively hydrophilic or hydrophobic 20

surfaces (Cui, Cheng, Li, Zhou, Zhang & Chang, 2012; Wang, Xie, Huang, Liang & Zhou, 2015). In summary, the biocompatibility of KGM was effectively improved by acetylation. Macrophages play a crucial role in inflammatory responses, cytokine release and the regulation of somatic cell behaviors during wound healing (Kloc, Ghobrial, Wosik, Lewicka, Lewicki & Kubiak, 2019; Wynn & Vannella, 2016). KGM is an established macromolecular ligand with a mannose receptor and has been demonstrated to activate macrophages to secrete pro-angiogenic/pro-mitogenic GFs, which induce

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blood vessel formation in vivo (Feng et al., 2017). In this study, a series of experiments were designed to verify the immunomodulation of AceKGM. First, the cell adherence rate suggested that there was a specific interaction between AceKGM

and the M2 phenotype Raw264.7 cells, and the adhesive force was quantitatively

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detected by AFM-SCFS. This result proved that there was a higher adhesive force

between the M2 phenotype Raw264.7 cells and the AceKGM-1.0 surface than

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between the other cells and this surface. Second, the ELISA results indicated that AceKGM up-regulated the M2 phenotype in Raw264.7 cells expressing

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anti-inflammatory/pro-regenerative cytokines. These outcomes were consistent with several reports in that the mannose units in KGM could be recognized by the MR on

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M2 phenotype Raw264.7 cells (Luo, Diao, Xia, Dong, Chen & Zhang, 2010; Niu et al., 2017). Moreover, the M2 phenotype Raw264.7 cells expressed more anti-inflammatory/pro-regenerative cytokines on the AceKGM-1.0 fibrous membrane

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than on the other membranes, which served as a proof-of-concept that appropriate hydrogen bonding is beneficial to the recognition between the mannose receptor and

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AceKGM (Unno et al., 2016). This result proved that chemical compositions are crucial for polysaccharide biological functions. In brief, AceKGM fibrous membranes were

confirmed

to

promote

M2

phenotype

Raw264.7

cells

to

express

anti-inflammatory/pro-regenerative cytokines by a specific interaction between the mannose units of KGM and the MR of M2 Raw264.7 cells. The detailed mechanism underlying this bioactivity would require further studies, while these results warranted that the AceKGM-based scaffold has the potential to promote wound healing in vivo. 21

AceKGM could be electrospun into a fibrous membrane with certain mechanical properties that is stable in a physiological solution but could not meet the basic requirements for a wound dressing with a slow degradation rate. In this study, type I collagen was co-electrospun with AceKGM-1.0 to obtain a fibrous membrane with fast degradation and good bioactivity. The in vivo experiments proved that AceKGM decreased the inflammatory response and promoted wound closure. The reasons were that AceKGM could reduce the release of inflammatory cytokines at the early stages and could stimulate M2 macrophages expressing IL-10 and TGF-β1 for the

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recruitment and proliferation of fibroblasts via the fibroblast growth factor 2/ERK pathway (Li, Yan, Yang, Ying, Hui & Zhenhua, 2012), which resulted in rapid

collagen synthesis and epithelialization. This study offers a new strategy to harness

the power of host innate immunity with engineering materials, which could improve

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tissue regeneration in a more physiologically relevant way.

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5. Conclusions KGM was acetylated with TFAA and acetic acid with different DS, and its hydrophobicity and bioactivity were significantly improved. AceKGM adhered M2 phenotype macrophages more effectively and induced them to express abundant anti-inflammatory and pro-regenerative cytokines. AceKGM or its mixture with type I collagen could be electrospun into a fibrous membrane. The in vivo assessment showed that the A/C55 fibrous membrane efficiently activated macrophages and accelerated wound healing in mice by promoting re-epithelialization, tissue

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remodeling, and collagen deposition. These results demonstrated that the AceKGM-based membranes have potential in skin wound regeneration as a bioactive

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scaffold.

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Acknowledgments This work was supported by research grants from the National Natural Science Foundation of China (31570964) and the Natural Science Foundation of Guangdong

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Province China (2016A030313854).

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