Fabrication of Antibacterial Collagen-Based Composite Wound

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Fabrication of Antibacterial Collagen-Based Composite Wound Dressing Liming Ge, Yongbin Xu, Xinying Li, Lun Yuan, Huan Tan, Defu Li, and Changdao Mu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01482 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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ACS Sustainable Chemistry & Engineering

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Fabrication of Antibacterial Collagen-Based Composite Wound

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Dressing

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Liming Ge,† Yongbin Xu,†,‡ Xinying Li,＃ Lun Yuan,† Huan Tan,§ Defu Li,*,† and Changdao Mu†

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†

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University, Chengdu 610065, P. R. China

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‡

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Baotou 014010, P. R. China

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Department of Pharmaceutics and Bioengineering, School of Chemical Engineering, Sichuan

School of Life Science and Technology, Inner Mongolia University of Science and Technology,

＃

College of Chemistry and Environment Protection Engineering, Southwest Minzu University,

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Chengdu 610041, P. R. China

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§

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Department, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610052, P. R.

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China

Key Laboratory of Medicinal and Edible Plants Resources, Development of Sichuan Education

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*Corresponding Author.

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E-mail address: [email protected] (D. Li).

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Address (all the authors): Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu

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610065, P. R. China

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Collagen is the favorable candidate in the field of biomaterials as wound dressings.

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However, it cannot be ignored that the application of collagen is limited to its poor physicochemical

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and perishable properties. It is significant to endow collagen with antibacterial activity and

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simultaneously improve the physicochemical properties. Here, we present a simple method to

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fabricate antibacterial collagen-based wound dressing with appropriate physicochemical properties.

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Firstly, dialdehyde xanthan gum (DXG) was used as environmental friendly reducing agent and

30

stabilizer to synthesize silver nanoparticles (AgNPs). Then, collagen/DXG-AgNPs composite

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dressings were fabricated by directly immersing collagen sponge in the obtained DXG-AgNPs

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aqueous solutions. Our results showed that the spherical AgNPs with diameters of 12~35 nm were

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successfully synthesized. The presence of DXG effectively prevented aggregation and precipitation

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of AgNPs in aqueous solution. By the simple one-step solution-immersion approach, AgNPs were

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homogeneously introduced into collagen matrix and collagen was simultaneously crosslinked by the

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existent DXG. The robust antibacterial activity was endowed to collagen as expected while the

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physicochemical properties of collagen were effectively improved. It is interesting that

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collagen/DXG-AgNPs composite dressings possessed functions of shape memory, good blood

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compatibility and cytocompatibility. In addition, collagen/DXG-AgNPs composite dressings could

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accelerate the deposition of collagen and thereby effectively promote full-thickness burns healing

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without scar formation.

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KEYWORDS: Biopolymers, Wound healing, Antibacterial activity, Dialdehyde xanthan gum,

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Silver nanoparticle, Green synthesis

45 46 47 48

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INTRODUCTION

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Bacterial infection is one of the most critical issues that delays the wound healing in burn injuries.1

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What’s more, the body dehydration also makes adverse effect on wound healing.2 Therefore, the

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biomaterial-based wound dressings have been extensively applied in protecting internal organs from

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the external environment when the skin is damaged.3-5 Wound dressings can be act as the effective

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physical barrier against exogenous microbial invasion and maintain the wound moist environment

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to promote the wound healing. 1, 4 Collagen, a primarily structural protein, is one of the most widely

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used tissue-derived materials with characteristic properties of unique biological functions, excellent

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biocompatibility, well biodegradability and weak antigenicity.6 Collagen is the favorable candidate

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in the field of biomaterials as tissue engineering scaffolds and wound dressings.7-8 However, it

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cannot be ignored that the application of pure collagen material is usually limited to its poor

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mechanical strength, low thermal stability, weak water resistance, rapid biodegradation and

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perishable property. Many ways have been applied to overcome the above mentioned problems of

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collagen, such as crosslinking, adding antimicrobial agents and compounding with nanoparticles.5,

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9-13

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Silver nanoparticles (AgNPs) have gained extensive attention in recent years owing to their

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robust antimicrobial activity towards a wide range of microorganisms, including bacterial and

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fungal species.14-16 Moreover, AgNPs are considered to be the mostly effective agent to deal with

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the problem of multidrug-resistant (MDR) microorganisms for current medicine.1, 3, 16 For these

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reasons, AgNPs have been extensively utilized in the fields of antimicrobials and biomedical

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materials.1,

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Comparatively speaking, chemical reduction is the more time saving, easily controllable and

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effective method to synthesize AgNPs. Hydrazine,18 N, N-dimethyl formamide,19 sodium

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borohydride 20 and surfactants 21 are the common organic reducing agents, which have been used to

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fabricate the shapes and sizes controllable AgNPs. However, it is highly difficult to remove the

4-5, 17

Physical and chemical methods are usually used to fabricate AgNPs.

3



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toxic and harmful compounds in the reduction system. So these produced AgNPs are not suitable

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for biomedical application. Tollens reaction is the common route to synthesize low toxic AgNPs

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with controlled size.22 But the added aqueous ammonia, used to form tollens reagent [Ag(NH3)2]+,

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also has potential biological risk. Therefore, it is urgently needed to develop green and

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environmentally friendly approaches for the fabrication of riskless AgNPs for biomedical

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application. The natural derived reducing agents, such as citric acid,23 ascorbic acid,24 glucose,25

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histidine,5 heparin,26 dopamine17 and polysaccharides22, 27-29 are the safe, eco-friendly and promising

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choices, which can reduce Ag+ to Ag0 by directly mixing with silver source under certain conditions.

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Unfortunately, AgNPs chemical reduced by the reducing agents with low molecular weight tend to

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aggregate.25,

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aqueous solution. Thereby the extra dispersant polyvinyl pyrrolidone (PVP) was added to overcome

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the drawback.25 It was reported that AgNPs reduced by xylan via Tollens reaction were well

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dispersed and stable in aqueous solution.22 AgNPs reduced by locust bean gum were highly stable

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over 7 months.31 Moreover, AgNPs were stable in chitosan solution till several months, resulting

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from that chitosan can act as stabilizing ligand to prevent colloidal instability.24 To sum up,

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polysaccharides are the promising and environmental friendly candidates to synthesize and stabilize

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AgNPs for biomedical application.

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For example, AgNPs reduced by glucose would rapidly aggregate in resulting

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Nowadays, combining nanoparticles with organic components to fabricate nanohybrid materials

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is attracting significant attention.32 Collagen/AgNPs nanohybrid materials have been prepared in

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previous works. AgNPs successfully endowed collagen with good antibacterial properties against

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both Gram positive and Gram negative bacteria.5, 33 However, AgNPs showed limited contribution

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to the improvement of physicochemical properties of collagen.5 In addition, the homogeneous

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distribution of AgNPs in collagen matrix is meaningful but of great challenge. Remarkably, it is of

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great significance to develop a simple method to homogeneously introduce AgNPs into collagen

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matrix and simultaneously improve the physicochemical properties of collagen. 4


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In our previous work, we found that dialdehyde polysaccharide as the effective crosslinking agent

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can be used for collagen fixation with good biocompatibility and low cytotoxicity.9, 12 In this work,

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dialdehyde xanthan gum (DXG) was firstly chosen as environmental friendly reducing agent and

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stabilizer to synthesize AgNPs under heating in the presence of silver nitrate. Afterwards, collagen

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sponge was directly immersed into the homogeneous DXG-AgNPs composite solution to obtain

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collagen/DXG-AgNPs composite wound dressings. By the simple one-step solution-immersion

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approach, AgNPs were uniformly introduced into collagen matrix meanwhile collagen was

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crosslinked by the existent DXG. Collagen was endowed with antibacterial properties while its

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physicochemical properties were simultaneously improved through above process. The reaction

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conditions and structural properties of DXG-AgNPs composite were characterized. The

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physicochemical and biological properties of collagen/DXG-AgNPs composite dressings were

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systematically investigated with microstructure, crosslinking degree, porosity, swelling property,

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moisture retention capacity, water vapor transmission rate, mechanical properties, antibacterial

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activity, in vitro cytotoxicity and blood compatibility. In vivo animal experiment was further

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preformed to evaluate the actual full-thickness burn healing effect of the as-fabricated composite

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wound dressings.

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MATERIALS AND METHODS

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Materials. Xanthan gum, sodium periodate, silver nitrate, bovine serum albumin (BSA) and

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Sirius Rose BB were purchased from Aladdin Reagent Database Inc. (Shanghai, China). Bovine

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achilles tendon was obtained from Regional Slaughter House (Chengdu, China). Sulfuric acid,

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sodium sulfide, acetone, acetic acid, nitric acid, 30% hydrogen peroxide solution, anhydrous

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ethanol and ninhydrin were purchased from Kelong Chemical Reagent Company (Chengdu, China).

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All chemicals were used as received unless indicated otherwise. The bacteria strains of Escherichia

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coli (E. coli, ATCC 8739), Staphylococcus aureus (S. aureus, ATCC 6538) and Pseudomonas 5



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aeruginosa (P. aeruginosa, ATCC 9027) were purchased from China Center of Industrial Culture

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Collection (Beijing, China). The mouse fibroblast cells line (L929) was provided by the

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Engineering Research Center in Biomaterials of Sichuan University. Dulbecco’s modified Eagle’s

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medium (DMEM), benzylpenicillin 100 IU/mL, streptomycin 100 IU/mL, trypsin, fetal bovine

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serum (FBS) and phosphate buffer solution (PBS) were purchased from Hyclone (Thermo Fisher,

129

USA).

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4-disulfophenyl)-2H-tetrazolium] monosodium salt (CCK-8) was purchased from Dojindo

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Laboratories (Kumamoto, Japan). White rabbits were purchased from Chengdu Dashuo Laboratory

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Animal Co., Ltd (Chengdu, China).

Tetrazolium-8-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,

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Preparation of DXG-AgNPs Composite. Dialdehyde xanthan gum (DXG) with about 22.5%

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aldehyde content was fabricated by periodate oxidation according to our previous work.34 DXG was

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dissolved in deionized water with the concentration of 10 mg/mL and stirred for 24 h at room

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temperature. Then aqueous solution of silver nitrate with the concentration of 10 mg/mL was added

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to above DXG solution and stirred for 20 min at room temperature in dark. The mixture was then

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heated to boiling (100 oC) for designed time with the method of reflux condensation. After that, the

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resulting solution was cooled to room temperature under stirring. The solution was dialyzed for 5

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days and then deionized water was added up to 25 mL. Finally, the above solution was centrifuged

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at 6000 r/min for 10 min, and the supernatant was collected as sample. The detailed information

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about the different reaction conditions are listed in Table S1.

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Characterization of DXG-AgNPs composite. UV-Vis absorption of DXG-AgNPs composite

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solution was recorded using an UV-Vis spectrophotometer (Alpha-1860, Shanghai Lab-Spectrum

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Instruments Co., Ltd, China) with a scan range of 300~800 nm. The morphology of DXG-AgNPs

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composite was observed by transmission electron microscope (TEM, Libra 200FE, Zeiss, Germany)

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equipped with energy dispersive X-ray spectroscopy (EDS). Size distribution of DXG-AgNPs was

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measured on a zetasizer nano-ZS ZEN3600 instrument (Malvern, England). Chemical composition 6


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of DXG-AgNPs composite was determined by X-ray photoelectron spectroscopy (XPS, ESCALAB

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250Xi, Thermo Scientific, USA) equipped with monochromated Al Kα radiation (hν=1486.6 eV).

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X-ray diffraction (XRD) of DXG and DXG-AgNPs composite were measured using a X-ray

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diffractometer (D8 ADVANCE, Bruker, Germany) equipped with a Ni-filtered CuKα radiation

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source (λ=0.154 nm). The operating voltage was set as 18 kV and the range of diffraction angle was

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set as 5°~90°.

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Preparation of Collagen/DXG-AgNPs Composite Dressings. Collagen type I was isolated

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from fresh adult bovine archilles tendon according to previous method.35 The 10 g of collagen

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aqueous solution (0.5%, w/w) were poured into iron box (5×5 cm) and subsequently freeze-dried to

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obtain collagen sponge (Col). Then the collagen sponge was directly soaked into original and 2-fold

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diluted DXG-AgNPs composite solutions for 30 mins at room temperature. The obtained

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collagen/DXG-AgNPs composite dressings were collected, washed with deionized water and

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freeze-dried. The collagen/DXG-AgNPs composite dressings were labeled as Col-Ag1 and Col-Ag2

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when 2-fold diluted and original DXG-AgNPs solutions were used, respectively.

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Microstructure Observation. The microstructure of collagen/DXG-AgNPs composite dressings

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was recorded using a field emission SEM instrument (JEOL JSM-7500F, Japan). The surface of

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samples was coated with Au before imaging and the operating voltage was set as 5 kV. In addition,

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the collagen/DXG-AgNPs composite dressings were embedded by epoxy resin and then frozen

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sectioned by freezing-microtome (EM UC7, Leica, Germany). The frozen section of samples was

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observed by TEM to check the appearance of AgNPs.

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Measurement of Silver Content. The 0.03 g of dry collagen/DXG-AgNPs composite dressings

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was added to a solution containing 10 mL of nitric acid and 5 mL of hydrogen peroxide (30%, w/v).

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Then the solution was heated until transparent. After that, the solution was topped with deionized

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water up to 10 mL. The silver concentration of the solution was determined by inductively coupled

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plasma-optical emission spectrometry (ICP-OES, OPTIMA 8000, PerkinElmer, USA). The silver 7



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content of sample was calculated by the following equation:

Silver Content ( mg / g ) =

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C ×V m

(1)

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where C is the silver concentration (mg/mL) of measured solution calculated from the standard

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curve, V is the volume (mL) of measured solution and m is the weight (g) of sample. Three samples

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were measured for each type of product.

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Measurement of Crosslinking Degree. Ninhydrin assay was used to determine the crosslinking

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degree of samples.13 Briefly, 0.1 g of dry sample, 1 mL of solution type A, 1 mL of solution type B

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and 8 mL of distilled water were blended and heated boiling for 20 mins. After cooling to room

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temperature,

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spectrophotometer at 570 nm, which was used to calculate the amino concentration of resulting

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solution according to standard curve. Glycine solutions at a series of concentrations were used to

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make standard curve. Solution type A was obtained by dissolving 2.1 g of citric acid, 0.8 g of

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sodium hydroxide and 0.08 g of stannous chloride into 100 mL of distilled water. Solution type B

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was prepared by dissolving 4 g of ninhydrin in 100 mL of ethylene glycol monomethyl ether. The

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crosslinking degree of samples was calculated by the following equation:

the

optical

absorbance

of

resulting

Crosslinking Degree ( % ) =

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solution

C2 − C1 ×100 C2

was

recorded

by

UV-Vis

(2)

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where C1 is the amino concentration of aqueous solution of hybrid materials while C2 is that of

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collagen. Each measurement was repeated three times and the average value was taken as the final

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result.

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Measurement of Porosity. The weight (m1) and volume (V) of samples were recorded. Then the

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samples were immersed in anhydrous ethanol under stirring. After swelling to equilibrium, the

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weight (m2) of swollen samples was measured. The porosity of samples was calculated by the

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following equation:

8


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Porosity ( % ) =

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m2 − m1 × 100 ρV

(3)

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where ρ is the density of anhydrous ethanol. The measurement was repeated for five times of each

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sample.

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Swelling Characteristics and Moisture Retention Capacity. The initial weight of rounded

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samples with a diameter of 30 mm was weighed (m0). Then samples were immersed in distilled

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water and swollen for 2 h at room temperature. After that, the swollen samples were taken out and

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reweighed (mt) after gently blotting with filter paper. The swelling ratio (SR) of samples was

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calculated by the following equation:

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SR (%) =

mt − m0 × 100 m0

(4)

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The measurement was repeated for five times of each sample. Then the samples were placed in

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an incubator at 37 oC and 50±2% RH. The SR values of samples were measured every 1 h to

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evaluate their moisture retention capacity.

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Measurement of Water Vapor Transmission Rate. Water vapor transmission rate (WVTR) of

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samples was measured based on the method of American Society for Testing and Materials (ASTM)

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E96-90, procedure D.36 Samples were cut into disk with a diameter of 30 mm and sealed on the top

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of glass permeating cups containing 20 mL of deionized water, which were then placed in an

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incubator at 37 oC and 50±2% RH. The weight of cups was recorded every 1 h till to 10 h periods.

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The WVTR values of samples were calculated using following equation:

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WVTR [(g/(m 2 × d)] =

w × 24 A×t

(5)

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where w is the weight loss (g) during the period of time t (h), A is the area of exposed sample (m2).

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The whole measurement was repeated five times for each sample.

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Measurements of Mechanical Properties. Dry samples were swollen in distilled water for 2 h at

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room temperature to get swollen samples. Each dry and swollen sample was cut into dumbbell-like 9



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strips. Then tensile strength (TS) and elongation at break (EB) of dry and swollen samples were

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measured according to the standard testing method ASTM D882-97 using a microcomputer control

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electronic universal testing machine (CMT6202, MTS systems Co., LTD., China). The crosshead

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speed was set at 20 mm/min. TS (MPa) and EB (%) of samples were calculated using the following

224

equations:

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TS (MPa) = Fmax / A

(6)

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EB (%) = ( L / 20) × 100

(7)

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where A is the area (m2) of cross-section at where the sample rupture. Fmax and L are the maximum

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force load (N) and real length of elongation (mm) of the sample at the moment of rupture,

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respectively. 20 is the initial narrow length (mm) of dumbbell-like strips before test. Ten pieces of

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each sample were tested and the average value was taken as the final result.

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Antibacterial Activity Evaluation. Inhibition Zone Method. Inhibition zone method was used as

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semi-quantitative test to evaluate the antibacterial property of samples. Generally, 100 µL of E. coli,

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S. aureus and P aeruginosa suspension (109 CFU/mL) were uniformly spread on Luria-Bertani (LB)

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agar plates, respectively. Then the rounded samples (15 mm in diameter) after ultraviolet

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sterilization were gently placed on the surface of LB agar and incubated at 37 oC for 24 h. The

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bacterial-free zones surrounding the rounded samples were measured and used to evaluate the

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antibacterial capacity of samples. The tests were repeated for three times.

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Bacterial Infiltration. Bacterial infiltration through samples was studied to evaluate the

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antibacterial property of samples again. The 2×2 cm2 square samples after ultraviolet sterilization

240

were placed on the surface of LB agar. Then 100 µL of E. coli, S. aureus and P aeruginosa

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suspension (109 CFU/mL) were dropped on the surface of samples, respectively, and incubated at

242

37 °C for 24 h. The growth of bacteria on the surface of samples was recorded by camera. Moreover,

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the samples containing bacteria were collected and fixed with 2.5% (v/v) glutaraldehyde for 2 h at

244

4 °C. After that, the samples were washed with phosphate buffered saline (PBS, pH=7.4) and 10


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dehydrated using a graded series of ethanol following by freeze-drying. The morphologies of

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bacteria on the surface of samples were observed by SEM after coating with Au. Furthermore, the

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LB agar under the samples was taken out and put into 25 mL of sterile water to obtain bacterial

248

suspension. Then 100 µL of above bacterial suspension were uniformly spread on new LB agar

249

plates and incubated at 37 °C for 24 h again. The growth of bacteria was recorded to further detect

250

the number of bacteria infiltrating through the samples.

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In Vitro Blood Compatibility. Bovine Serum Albumin (BSA) Adsorption. Adsorption of BSA on

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collagen sponge and collagen/DXG-AgNPs composite dressings was studied to understand the

253

interaction between blood protein and our materials.37 BSA was dissolved in PBS (0.5 M, pH=7.4)

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with the concentration of 2 mg/mL. Samples were swollen in PBS (pH=7.4) for 2 h and then

255

weighed after removing the surface liquid. Subsequently, the swollen sample was immersed in 20

256

mL of BSA solution and incubated for 30 min at 37 °C under stirring. Then the optical absorbance

257

of BSA solution was recorded by UV-Vis spectrophotometer at 278 nm, which was used to calculate

258

the concentration of BSA solution based on standard curve. BSA solutions with different

259

concentrations were used to make standard curve. The amount of BSA adsorbed on samples was

260

calculated using following equation:

261

Absorbed BSA (mg/g ) =

2 − Ca × 20 W

(8)

262

where Ca is the concentration of BSA solution (mg/mL) after adsorption, W is the weight (g) of

263

swollen sample. The whole measurement was repeated five times for each sample.

264

Whole-Blood Dynamic Clotting Study. Kinetic clotting time method was used to investigate the

265

blood clotting ability of samples according to literature.38 The 5.0 mL of fresh blood drawn from

266

healthy rabbit, 1.0 mL of anticoagulant citrate dextrose (ACD) solution and 6.0 mL of 0.9% (w/v)

267

sodium chloride aqueous solution were mixed to obtain ACD blood. Samples after ultraviolet

268

sterilization were swollen in sterile water for 2 h. Then 200 µL of ACD blood were dropped on the

269

surface of swollen sample. Subsequently, 25 µL of CaCl2 solution at 0.2 mol/L were added and 11



Page 12 of 42

270

mixed well in the ACD blood to initiate clotting. The clotting was performed for a predetermined

271

period of time at 37 °C. Then samples were transferred into 100 mL of deionized water. After

272

incubation at 37 °C for 10 min, red blood cells which were not trapped in the clot would

273

haemolyzed fully. The optical absorbance of resulting solution was recorded by UV-Vis

274

spectrophotometer at 545 nm. The control group was set as directly adding 200 µL of ACD blood in

275

100 mL of deionized water and incubated at 37 °C for 10 min. The blood clotting ability of glass

276

was also tested in the experiment. Blood clotting index (BCI) can be calculated by the following

277

equation:

BCI (%) =

278

At ×100 A0

(9)

279

where At and A0 are the absorbance values of test group and control group, respectively. Five times

280

of test were repeated for each sample.

281

In Vitro Cytotoxicity Studies. The viability of L929 fibroblasts was determined by CCK-8 assay

282

to evaluate the in vitro cytotoxicity of fabricated samples.4, 39 Samples after ultraviolet sterilization

283

were immersed in DMEM containing 10% fetal bovine serum and 1% antibiotic/antimycotic

284

solution and incubated at 37 °C for 24 h. Then samples were removed to obtain extraction liquid.

285

The 100 µL of DMEM containing L929 fibroblasts were added to 96-well plates (2×103 cells/well)

286

and incubated at 37 °C, 5% CO2 for 24 h. Then the DMEM culture medium was replaced by 100 µL

287

of extraction liquid. After 1, 2, and 3 days of incubation, 100 µL of fresh DMEM supplemented

288

with CCK-8 (10%, v/v) were used to replace extraction liquid and further incubated for 90 min.

289

Then the absorbance values of culture media were recorded with a microplate reader (ELX 800,

290

BioTek Instruments Inc., USA) at 450 nm. DMEM culture medium only with cells was set as

291

control group. DMEM culture medium containing CCK-8 (10%, v/v) without cells was set as blank

292

group. The cell viability was calculated by the following equation:

293

Cell Viability (%) =

As − Ab × 100 Ac − Ab 12


(10)

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294

where As, Ac and Ab are the absorbance values of experimental, control and blank groups,

295

respectively. The experiment was repeated five times for each sample.

296

In Vivo Animal Experiment. The wound healing characteristics of various as-fabricated samples

297

were evaluated using a rabbit model. All the protocols for animal experiments were performed

298

according to the guidelines of the Council for the Purpose of Control and Supervision of

299

Experiments on Animals, Ministry of Public Health, China. White rabbits, about 2 kg of weight of

300

each, were employed to evaluate wound healing characteristics. All the rabbits were anesthetized by

301

the intraperitoneal injection of pentobarbital sodium (Sigma-Aldrich) at a dose of 30 mg/kg. Then,

302

the dorsal hair of rabbits was denuded with 8% (w/v) Na2S aqueous solution. Afterwards, the burns

303

on both sides of the dorsum of each rabbit were made using a hot circular copper billet (20 mm in

304

diameter). The burns were excised to the level of the panniculus carnosus to create full-thickness

305

burns on the back of each rabbit. Then, 100 mL of P. aeruginosa suspension (109 CFU/mL) were

306

uniformly spread on each wound area. After 2 h of bacterial challenge to the full-thickness burns,

307

the wounds were covered with samples firstly and then wrapped with sterile gauze and fixed with

308

an elastic bandage. The covered samples were replaced at every 2 days. The full-thickness burns

309

covered with sterile gauze only were set as control group. The wound healing progress of the

310

full-thickness burns was recorded at different time. For histological examination, the rabbits were

311

sacrificed at the day 18. The regenerated skin tissues of rabbits were excised and fixed with 10%

312

formalin. Then, the skin tissue was frozen sectioned using freezing-microtome (EM UC7, Leica,

313

Germany) before staining with Sirius Rose BB. Finally, the obtained sections were observed under

314

an optical microscope (CKX53, Olympus, Japan).

315 316

RESULTS AND DISCUSSION

317

Synthesis and Characterization of DXG-AgNPs. Polysaccharides, mainly on account of the

318

existent plenty of hydroxyl groups, can reduce Ag+ to AgNPs as reported.27, 30, 40 Here, the method 13



319

using dialdehyde xanthan gum (DXG) as environmental friendly reducing agent and stabilizer was

320

applied to synthesize AgNPs. The UV-Vis absorption spectra of AgNO3 and DXG-AgNPs aqueous

321

solutions with different ratios of AgNO3 to DXG are shown in Figure 1A. It can be seen clearly that

322

AgNO3 aqueous solution does not show any absorption at wavelength range of 350~800 nm.

323

However, a new absorption peak at around 420 nm comes out after boiling the mixed aqueous

324

solutions of DXG and AgNO3. This new peak is caused by the typical surface plasmon resonance

325

(SPR) of Ag nanoparticles (AgNPs).24, 41 The result suggests that AgNPs are formed in the reduction

326

system of DXG/AgNO3 mixed aqueous solutions under heating. Note that the intensity of SPR

327

peaks tends to gradually increase with the increasing dosage of DXG. Meanwhile, the color of

328

DXG-AgNPs solutions was getting deeper and deeper as showed in inset of Figure 1A. It indicates

329

that more AgNPs are generated in DXG-AgNPs aqueous solutions with the increasing dosage of

330

DXG. The time dependence of UV-Vis absorbance of DXG/AgNO3 solutions with 1:2 mass ratio of

331

AgNO3 to DXG is presented in Figure 1B. The results show that the intensity of SPR peaks keeps

332

increasing and the color of DXG/AgNO3 solutions changed from light yellow to brown with the

333

prolonged reaction time, indicating that more AgNPs are generated. However, AgNPs tended to

334

aggregate obviously to form larger particles when the reaction time is longer than 30 min (Figure

335

S1). Therefore, in view of the content and particle size of AgNPs, an optimal scheme to synthesize

336

AgNPs is boiling the mixture with a 1:2 mass ratio of AgNO3 to DXG for 30 min.

337

338 14


Page 14 of 42

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339 340

Figure 1. (A) UV-Vis absorption spectra of AgNO3 and DXG/AgNO3 solutions with different mass

341

ratios of AgNO3 to DXG reacted for 10 min and (B) Time dependence of UV-Vis absorbance of

342

DXG/AgNO3 mixed solutions with 1:2 mass ratio of AgNO3 to DXG. The insets of 1A and 1B

343

show the photographs of AgNO3 and DXG/AgNO3 solutions.

344

345 346

Figure 2. (A) XRD patterns and (B) wide scan XPS spectra of DXG and DXG-AgNPs composite.

347

(C) High-resolution XPS spectra of Ag 3d of DXG-AgNPs. (D) TEM image of DXG-AgNPs and (E)

348

the corresponding energy dispersive X-ray spectroscopy (EDS) profile. (F) High resolution

349

transmission electron microscopy (HRTEM) image and (G) selected-area electron diffraction 15



Page 16 of 42

350

(SAED) pattern of single AgNPs. The measured DXG-AgNPs was synthesized with 1:2 mass ratio

351

of AgNO3 to DXG and reacted for 30 min.

352 353

The XRD patterns of DXG and DXG-AgNPs composite are showed in Figure 2A. The broad 34

354

diffraction peak at around 20.3° is assigned to the characteristic XRD peak of DXG.

Note that

355

new characteristic peaks at 2θ=38.4°, 44.6°, 64.6°, 77.5° and 81.7° are observed in DXG-AgNPs

356

composite, which correspond to the (111), (200), (220), (311) and (222) facets of metallic Ag,

357

respectively.4-5, 22 The results indicate that the crystal silver was successfully synthesized in this

358

reduction system. X-ray photoelectron spectroscopy (XPS) spectra of DXG and DXG-AgNPs

359

composite are showed in Figure 2B. It shows that the C, O and Ag elements are detected in

360

DXG-AgNPs composites. The XPS peaks for C and O are assigned to DXG, while the new XPS

361

peaks are ascribed to the Ag 3d and Ag 3p binding energies of synthesized AgNPs. Figure 2C shows

362

the high-resolution XPS spectra of Ag 3d of DXG-AgNPs composites. Two individual peaks are

363

observed at binding energies of 369.6 eV and 375.6 eV, which are attributed to Ag 3d5/2 and Ag

364

3d3/2, individually.42 The difference value of binding energy of Ag 3d5/2 and Ag 3d3/2 is 6.0 eV,

365

indicating the natural characteristic of metallic silver.22 However, the XPS peaks here are slightly

366

larger than that of pure metallic Ag (Ag 3d5/2, 367.4 eV and Ag 3d3/2, 373.4 eV).43 It is mostly on

367

account of the interactions between AgNPs and DXG. The same result was reported in previous

368

study.22 Therefore, it can be inferred that the synthesized AgNPs are capped by DXG.

369

The successful synthesis of AgNPs was further confirmed by transmission electron microscope

370

(TEM) and the result is showed in Figure 2D. It shows that AgNPs present spherical particles with

371

diameters of 12-35 nm. The corresponding energy dispersive X-ray spectroscopy (EDS) profile

372

further confirms the presence of Ag. Note that Figure 2E shows C and O signals besides Ag signal.

373

It suggests that the synthesized AgNPs are capped by DXG. The same result was also observed

374

when xylan was used as reducing agent to fabricate AgNPs.22 The signals of Cu are due to the 16


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375

copper mesh used to hold samples during the measurements. Figure 2F shows that the interplanar

376

distance of AgNPs is about 0.23 nm, which matches well with the (111) plane of typical pure

377

metallic Ag (JCPDS file no: 89-3722). The selected-area electron diffraction (SAED) pattern of

378

single AgNPs is showed in Figure 2G, which reveals that synthesized AgNPs possess

379

monocrystalline nature.

380

381 382

Figure 3. (A) UV-Vis absorption spectra of DXG-AgNPs (30 min) solution stored for different days.

383

The inset of A is the photographs of DXG-AgNPs solution (30 min) at day 1 and day 180. (B) Size

384

distribution of synthesized DXG-AgNPs (30 min) in aqueous solution.

385

386

The UV-Vis absorption spectra of DXG-AgNPs aqueous solution stored for different days were

387

recorded to evaluate the stability of DXG-AgNPs aqueous solution, and the results are presented in 17



388

Figure 3A. It shows that the UV-Vis absorption spectra nearly keep the same and the intensity of

389

SPR peaks slightly decreases from day 1 up to day 180. Moreover, the color of DXG-AgNPs

390

solution is slightly changed and precipitation is not found as showed in the inset of Figure 3A. The

391

results indicate that the DXG-AgNPs aqueous solution is quite stable. The size distribution of

392

DXG-AgNPs in aqueous solution was measured by dynamic laser scattering (DLS) and the result is

393

showed in Figure 3B. The result shows that the average size of DXG-AgNPs is 422.9 nm. Note that

394

the average size of DXG-AgNPs in aqueous solution is much larger than that of AgNPs measured

395

by TEM. The results suggest that AgNPs and DXG are combined together and formed aggregates in

396

aqueous solution. The hydroxyl groups of DXG with high electronegativity property play an

397

important role in capping AgNPs due to their strong interactions.44 As is known to all, AgNPs trend

398

to aggregate easily in aqueous solution. Therefore, DXG capping can effectively improve the

399

stability of AgNPs in aqueous solution, which is beneficial to maintain their robust antibacterial

400

properties.45-46

401 402

Preparation and Structural Characterization of Collagen/DXG-AgNPs Composite

403

Dressings. Collagen/DXG-AgNPs composite dressings were fabricated by immersing pure collagen

404

sponge in DXG-AgNPs aqueous solutions and subsequently freeze-drying. By the simple one-step

405

solution-immersion approach, AgNPs and DXG could homogeneously diffuse into the

406

interconnected micropore of swollen collagen sponge. AgNPs could be physically bounded onto

407

collagen molecules due to the aminophilic nature of AgNPs.23 In addition, the crosslinking between

408

DXG and collagen would be formed through Schiff’s base reaction, which would make the collagen

409

sponge compact and restrict AgNPs in collagen matrix. Figure 4A shows the photographs of

410

collagen sponge (Col), DXG-AgNPs solution and collagen/DXG-AgNPs composite dressings. It

411

shows that collagen/DXG-AgNPs composite dressings present brownish yellow. The color of 18


Page 18 of 42

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412

Col-Ag2 is deeper than that of Col-Ag1 due to the higher concentration of DXG-AgNPs aqueous

413

solution used for fabrication of Col-Ag2. Figure 4B shows that collagen sponge and

414

collagen/DXG-AgNPs composite dressings present heterogeneous interconnected microporous

415

structure. However, the size of interconnected micropore of collagen/DXG-AgNPs composite

416

dressings is decreased due to the crosslinking effects of DXG. Figure 4C shows the TEM image of

417

Col-Ag2. It is clear that AgNPs are evenly introduced into collagen matrix as indicated by white

418

arrows through the simple one-step solution-immersion approach.

419 420

Figure 4. (A) Photographs of collagen sponge (Col), DXG-AgNPs solution (30 min),

421

collagen/DXG-AgNPs composite dressings. (B) SEM images of Col, Col-Ag1 and Col-Ag2 and (C)

422

TEM image of Col-Ag2. Col-Ag1 was fabricated by immersing pure collagen sponge in 2-fold

423

diluted DXG-AgNPs solution while Col-Ag2 was fabricated using original DXG-AgNPs solution.

424

425

The silver contents of collagen/DXG-AgNPs composite dressings were measured by ICP-OES

426

and summarized in Table 1. It shows that the silver content of Col-Ag2 (6.57±0.21 mg/g) is almost

427

double as that of Col-Ag1 (3.31±0.13 mg/g). It is because that Col-Ag1 was fabricated in double

428

diluted DXG-AgNPs solution while Col-Ag2 was fabricated in original one. More AgNPs would

429

diffuse into collagen sponge in DXG-AgNPs solution with higher concentration. Moreover, the

430

results indicate that AgNPs can be easily introduced into collagen matrix by directly immersing 19



Page 20 of 42

431

collagen sponge in DXG-AgNPs aqueous solution. The silver content can be easily controlled by

432

adjusting the concentration of DXG-AgNPs aqueous solution. Aldehyde groups of existed DXG in

433

DXG-AgNPs aqueous solution can react with amino groups of collagen via Schiff’s base reaction.

434

Here, the crosslinking degrees of collagen/DXG-AgNPs composite dressings were measured and

435

showed in Table 1. It shows that the crosslinking degree of Col-Ag2 is higher than that of Col-Ag1

436

owing to the higher concentration of DXG-AgNPs solution used for fabrication of Col-Ag2. The

437

thickness, volume change rate and porosity of collagen sponge and collagen/DXG-AgNPs

438

composite dressings are presented in Table 1. Crosslinking and nanoparticles incorporation could

439

tighten the structure of collagen sponge.9-10 Therefore, the thickness and porosity of

440

collagen/DXG-AgNPs composite dressings are obviously decreased while their volume change

441

rates are significantly increased. Col-Ag2 possesses higher crosslinking degree, resulting in higher

442

volume change rate, lower thickness and porosity than that of Col-Ag1.

443 444

Table 1. Silver Content, Crosslinking Degree, Thickness, Volumetric Change Rate and Porosity of

445

Collagen sponge (Col) and Collagen/DXG-AgNPs Hybrid Materials silver content

crosslinking

thickness

volume change

porosity

(mg/g)

degree (%)

(mm)

rate* (%)

(%)

Col

--

--

3.35±0.02

--

83.19±2.10

Col-Ag1

3.31±0.13

41.16±1.34

2.08±0.03

36.47±3.98

67.58±1.23

Col-Ag2

6.57±0.21

67.99±1.60

1.77±0.02

54.36±4.73

59.79±1.92

sample

446

*

447

means the fabricated sample with lower volume.

Volume change rate was the percentage of changed volume to initial volume. The bigger value

20


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448 449

Physical and Mechanical Properties of Collagen/DXG-AgNPs Composite Dressings.

450

Swelling ratio and moisture retention capacity of collagen sponge and collagen/DXG-AgNPs

451

composite dressings are depicted in Figure 5A. It shows that collagen sponge and

452

collagen/DXG-AgNPs composite dressings possess high liquid absorption capability. The swelling

453

ratios of Col-Ag1 and Col-Ag2 are ~4900% and ~3700%, respectively, which are lower than that of

454

collagen sponge (~11000%) owing to the crosslinking effects of DXG. Figure 5A shows that the

455

moisture contents of collagen sponge and collagen/DXG-AgNPs composite dressings decrease

456

almost linearly with time going at 37 oC and 50±2% RH. Note that the slopes (K, by linear fitting)

457

of Col-Ag1 and Col-Ag2 are much lower than that of collagen sponge. It indicates that the moisture

458

retention capacity of Col-Ag1 and Col-Ag2 is better than that of collagen sponge. The water vapor

459

transmission rates (WVTR) of collagen sponge and collagen/DXG-AgNPs composite dressings are

460

illustrated in Figure 5B. The WVTR values of Col-Ag1 and Col-Ag2 are about 1965 g/(m2×d) and

461

1778 g/(m2×d), respectively, which are lower than that of collagen sponge. The result is due to the

462

compact and dense microstructure of collagen/DXG-AgNPs composite dressings, which can bound

463

water molecules and restrain the evaporation loss of free water molecules. It is recommended that

464

the ideal wound dressing with WVTR value lower than 2500 g/(m2×d) is able to protect the injured

465

skin from wound dehydration.36 Hence, the collagen/DXG-AgNPs composite dressings may be

466

suitable for the application of wound dressings, which can absorb exudates and keep wound in a

467

moist environment.

468

The tensile strength and elongation at break of collagen sponge and collagen/DXG-AgNPs

469

composite dressings at dry state and swollen state were measured and showed in Figure 5C and 5D.

470

The tensile strength values of dry Col-Ag1 and Col-Ag2 are 413 kPa and 512 kPa, respectively,

471

while it is 251 kPa for the dry pure collagen sponge. It reveals that the tensile strength of

472

collagen-based sponge is significantly improved by the modification of DXG crosslinking and 21



473

AgNPs incorporation. It is well known that pure collagen materials can be highly swollen in

474

aqueous solution. Generally, the tensile strength of swollen pure collagen material is too low to be

475

measured. It is worth noting that the tensile strength of Col-Ag1 and Col-Ag2 at swollen state reach

476

to 62 kPa and 112 kPa, respectively. The tensile strength of Col-Ag2 (67% crosslinking degree) at

477

swollen state is even higher than that of dry genipin crosslinked collagen sponge (72 kPa at 67%

478

crosslinking degree).47 In addition, the elongation at break of Col-Ag1 and Col-Ag2 at swollen state

479

are much higher than that at dry state, indicating preferable flexibility. It is on account of the large

480

amount of water molecules in swollen composite dressings, which act as plasticizer.48-49 The above

481

results indicate that DXG crosslinking and AgNPs incorporation can effectively improve the

482

mechanical properties of composite dressings at both dry and swollen states.

483

484 485

Figure 5. (A) Swelling ratio and moisture retention capacity, (B) water vapor transmission rate 22


Page 22 of 42

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486

(WVTR), (C) tensile strength and (D) elongation at break of collagen sponge (Col) and

487

collagen/DXG-AgNPs composite dressings.

488

489 490

Figure 6. (A) Initial state, swollen state and the state after physical pressing of collagen sponge

491

(Col) and Col-Ag2. (B) Shape recovery process of swollen Col and Col-Ag2 in water after physical

492

pressing.

493 494

Figure 6A shows the initial state, swollen state and the state after physical pressing of collagen

495

sponge and Col-Ag2. It shows that collagen sponge showed a gelatinous appearance after fully

496

swelling. Indeed, the swollen collagen sponge had very poor strength, which was too low to be 23



497

detected. In comparison, Col-Ag2 still maintained the initial morphology and held good toughness

498

after fully swelling. Moreover, the free water absorbed by collagen sponge cannot be removed by

499

physical pressing. Indeed, the swollen collagen sponge cannot withstand the physical pressing,

500

which was easily shattered by physical pressing. It is interesting that the free water absorbed by

501

Col-Ag2 was easily removed by physical pressing and Col-Ag2 can keep the complete integrity

502

after physical pressing. Figure 6B shows the shape recovery process of swollen collagen sponge and

503

Col-Ag2 in water after physical pressing. Obviously, Col-Ag2 was quickly swollen again and

504

stretched to initial swollen state in water within 8 s while collagen sponge did not show this ability.

505

Indeed, the processes of swelling, physical pressing and swelling again can be repeated for Col-Ag2.

506

That is to say, Col-Ag2 possesses function of shape memory. It is mainly on account of the

507

decreased hydrophilicity and increased stiffness of collagen/DXG-AgNPs composite dressings after

508

DXG crosslinking and AgNPs incorporation.

509 510

Antibacterial Activity of Collagen/DXG-AgNPs Composite Dressings. Protein based materials

511

are easily contaminated by the environmental microorganisms due to their highly hydrophilic and

512

nutritious properties. Therefore, the method of adding antimicrobial agents, such as gentamicin

513

sulfate,50 nisin,13 curcumin51 and metallic particles,5, 52-53 is frequently used to endow protein based

514

materials with antimicrobial activity. As is known, AgNPs are the commonly applied antibacterial

515

agent with robust, broad-spectrum and none drug resistance properties, which are extensively used

516

in the fields of water sterilization and biomedical materials.3-4, 15-16, 22 In this study, the antibacterial

517

activity of collagen sponge and collagen/DXG-AgNPs composite dressings against E. coli, S.

518

aureus and P. aeruginosa were firstly evaluated by inhibition zone method and the results are

519

showed in Figure 7A. It is clear that no inhibition zone was observed for collagen sponge toward E.

520

coli, S. aureus and P. aeruginosa. However, the bacterial-free zones surrounding rounded Col-Ag1

521

and Col-Ag2 were obviously observed. In addition, the size of bacterial-free zones of Col-Ag2 was 24


Page 24 of 42

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522

larger than that of Col-Ag1 due to the higher content of AgNPs. The results indicate that the

523

incorporation of AgNPs confers collagen with antibacterial activity against both Gram-positive and

524

Gram-negative bacteria.

525 526

Figure 7. (A) Antibacterial activity of collagen sponge (Col) and collagen/DXG-AgNPs composite

527

dressings against E. coli, S. aureus and P. aeruginosa evaluated by inhibition zone method. (B)

528

Bacterial infiltration through Col and collagen/DXG-AgNPs composite dressings within 24 h and

529

(C) photographs of colonies of infiltrated bacteria after inoculation and incubation on new LB agar

530

for 24 h again.

531 532

The results of bacterial infiltration through collagen sponge and collagen/DXG-AgNPs composite 25



533

dressings are displayed in Figure 7B. It was found that E. coli, S. aureus and P. aeruginosa were

534

grown well on the surface of collagen sponge. After incubation for 24 h, bacterial colonies were

535

even observed surrounding collagen sponge. Bacterial colonies under collagen sponge were also

536

clearly observed after removal of collagen sponge. On the contrary, no bacterial colony was

537

observed on the surface and edge of Col-Ag1 and Col-Ag2. No bacterial colony was observed under

538

Col-Ag1 and Col-Ag2 too. The results further confirm the antibacterial activity of

539

collagen/DXG-AgNPs composite dressings. To further understand the barrier ability of composite

540

dressings to bacteria, the LB agar under collagen sponge, Col-Ag1 and Col-Ag2 were taken out,

541

homogenized in sterile water and subsequently incubated on new LB agar plates again. The growth

542

of bacteria was recorded in Figure 7C to detect the number of bacteria infiltrating through the

543

samples. The results show that the colonies of infiltrated bacteria through collagen sponge were

544

fully spread over the LB agar. However, relatively few bacterial colonies were observed on the LB

545

plates for Col-Ag1 and Col-Ag2. Note that almost no bacteria passed through Col-Ag2. The results

546

indicate that collagen/DXG-AgNPs composite dressings possess outstanding infiltration resistance

547

to bacteria. Col-Ag2 with high silver content demonstrates better effective ability to prevent

548

bacterial infiltration. The compact and dense microstructure of collagen/DXG-AgNPs composite

549

dressings makes contribution to slow down bacterial infiltration. Furthermore, the introduced

550

AgNPs endow collagen materials with excellent capacity of killing bacteria.

551

The morphologies of E. coli, S. aureus and P. aeruginosa after incubation for 24 h on the surface

552

of collagen sponge and collagen/DXG-AgNPs composite dressings were observed by SEM and

553

presented in Figure 8. It shows that bacteria grown well on the surface of collagen sponge and

554

presented smooth and plump morphology. However, fewer microorganisms were observed on the

555

surface of Col-Ag1 and Col-Ag2. The small amount of bacteria stuck together and showed morbid

556

morphology with rough, flat and wrinkled surface and even total lysis. The results further support

557

the antimicrobial mechanism of AgNPs that AgNPs could destroy the cell membranes and finally 26


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558

result in cell lysis.54-57 The same results were also achieved after incubating bacteria with

559

composites containing AgNPs in previous works.3, 16, 23, 58-59

560

561 562

Figure 8. SEM images of E. coli, S. aureus and P. aeruginosa after growth on the surface of

563

collagen sponge (Col) and collagen/DXG-AgNPs composite dressings at 37 oC for 24 h.

564 565

In Vitro Blood Compatibility and Cell Viability. Plasma proteins once contact with and adhere

566

to foreign materials will activate coagulation factor Ⅶ in a short time and promote to activate

567

platelets, followed by facilitating thrombin formation and platelet aggregation to further accelerate

568

thrombosis.60-62 That is to say, protein adsorption is the first step to initiate thrombosis when

569

biomaterials directly contact with blood. Protein adsorption is an important index to evaluate the

570

blood compatibility of biomaterials. Here, the adsorption of bovine serum albumin (BSA) on 27



571

collagen sponge and collagen/DXG-AgNPs composite dressings was detected and the results are

572

showed in Figure 9A. The results show that the amount of BSA adsorbed by collagen sponge (26.4

573

mg/g) is obviously larger than that by Col-Ag1 (22.5 mg/g) and Col-Ag2 (18.1 mg/g). Generally,

574

the lower amount of adsorbed BSA clearly implies the better antithrombotic effect, indicating the

575

better blood compatibility. The results suggest that collagen/DXG-AgNPs composite dressings have

576

better blood compatibility than collagen. Then the in vitro blood dynamic clotting on collagen

577

sponge and collagen/DXG-AgNPs composite dressings were conducted compared to glass. As

578

showed in Figure 9B, the blood clotting index (BCI) of collagen sponge is 37.5% when contacting

579

with ACD blood for 5 min, which is lower than that of Col-Ag1 (42.7%), Col-Ag2 (45.4%) and

580

glass (60.6%). Moreover, the BCIs of all samples are gradually decreased with time going on,

581

which tend to become equilibrium at approximate 30 min. The larger BCI demonstrates longer

582

clotting time and better antithrombogenicity, indicating preferable blood compatibility for

583

biomaterials.63 The results confirm that collagen/DXG-AgNPs composite dressings have better

584

blood compatibility than collagen. It is known that pure collagen is able to promote blood clotting

585

due to its intact triple-helix structure.64 Crosslinking affords dense network microstructure and

586

changed surface charge of collagen sponge, resulting in low amount of adsorbed BSA and high

587

BCI.65 The same results have been reported for dialdehyde carboxymethyl cellulose (DCMC)

588

crosslinked gelatin-PEG fibers and collagen cryogels.9, 12

589

To evaluate the biocompatibility of collagen/DXG-AgNPs composite dressings, the in vitro

590

cytotoxicity towards the mouse fibroblast cells line (L929) was measured using CCK-8 assay.

591

Figure 9C shows the viability of L929 cells incubated in extraction liquid of collagen sponge and

592

collagen/DXG-AgNPs composite dressings at 1 to 3 days. As showed in Figure 9C, the viability of

593

L929 cells incubated in extraction liquid of collagen/DXG-AgNPs composite dressings is slightly

594

lower than that of collagen sponge. It is mainly caused by AgNPs in composite dressings. Previous

595

study revealed that AgNPs would result in some harmful effects to cell viability.3 However, the 28


Page 28 of 42

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596

viability of L929 cells incubated in extraction liquid of collagen sponge and collagen/DXG-AgNPs

597

composite dressings is larger than 85% at 1 to 3 days, indicating no significant cytotoxicity of the

598

measured materials.58,

599

without any differences with blank, as showed in Figure S2. The results indicate that both collagen

600

sponge and collagen/DXG-AgNPs composite dressings have no cytotoxicity towards L929 cells.

66

Moreover, L929 cells showed nearly normal fibroblast morphology

601

602

603

29



604 605

Figure 9. (A) Amount of bovine serum albumin (BSA) adsorbed by collagen sponge (Col) and

606

collagen/DXG-AgNPs composite dressings. (B) Blood-clotting indexes (BCI) of glass, Col and

607

collagen/DXG-AgNPs composite dressings determined by whole-blood dynamic clotting study at

608

37 oC. (C) Viability of L929 cells incubated in extraction liquid of Col and collagen/DXG-AgNPs

609

composite dressings at 1 to 3 days.

610 611

In Vivo Evaluation of Wound Healing. In vivo animal experiment is indispensable for

612

evaluating the actual wound healing effect of as-fabricated composite wound dressings, in addition

613

to antibacterial examination, blood compatibility test and cytocompatibility assay.4 The wound

614

healing progress of the full-thickness burns after treatment with sterile gauze, Col, Col-Ag1 and

615

Col-Ag2 was recorded at different time as showed in Figure 10A. It can be seen clearly that all the

616

wounds after treating by Col, Col-Ag1 and Col-Ag2 were well healed at day 18 compared with the

617

sterile gauze (Blank) treated wound. More importantly, the regenerated skin at full-thickness burns

618

treated with Col-Ag1 and Col-Ag2 was smooth and similar to normal skin without scar formation.

619

Furthermore, the collagen/DXG-AgNPs composite dressings were capable to quickly promote the

620

growth of new rabbit hair at the regenerated skin. In one word, collagen/DXG-AgNPs composite

621

dressings exhibit excellent wound healing ability for the skin tissue.

622

The newly generated fibroblasts with little collagen will replace the necrotic tissue when the skin

623

tissue is injured. Once the amount of collagen increases, the fibroblasts will further develop into 30


Page 30 of 42

Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


624

fibrocytes and thereby the wound executes healing process.3 Therefore, the collagen deposition is

625

the key indicator in the process of wound healing.67-68 Micrographs of Sirius Rose BB stained

626

histological sections are showed in Figure 10B, which are used to estimate the extent of collagen

627

deposition in wound healing process. After healing for 18 days, compared to Col-Ag1 and Col-Ag2,

628

the sterile gauze and Col treated wound tissue presented sparse and untidy collagen fibers and

629

obviously lack of collagen. The collagen deposition at the collagen/DXG-AgNPs composite

630

dressings treated wound was obviously thicker and denser than that of the sterile gauze and Col

631

treated wound. The results indicate that collagen/DXG-AgNPs composite dressings exhibit the

632

functions to effectively repair full-thickness burns and improve deposition of collagen fibers in

633

ordered arrangement.

634

31



635 636

Figure 10. (A) Photographs of wounds after treatment with sterile gauze (Blank), collagen sponge

637

(Col) and collagen/DXG-AgNPs composite dressings at different time. (B) Micrographs of wound

638

tissues stained with Sirius Rose BB. The high-magnification images of b1 that marked with black

639

box are presented in b2. In b2, the blank area as indicated by black arrows shows the lack of

640

collagen (where the collagen was stained into rose red).

641 642

CONCLUSIONS

643

In this study, AgNPs were successfully synthesized using dialdehyde xanthan gum (DXG) as

644

environmental friendly reducing agent and stabilizer. The results showed that AgNPs formed in the 32


Page 32 of 42

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645

reduction system of DXG/AgNO3 aqueous solution under heating. The spherical AgNPs with

646

diameters of 12~35 nm were successfully synthesized by adjusting the dosage of DXG and reaction

647

time. Moreover, the presence of DXG effectively prevented aggregation and precipitation of AgNPs

648

in aqueous solution. Afterwards, the DXG-AgNPs solutions were used to fabricate

649

collagen/DXG-AgNPs composite wound dressings by immersing method. The results showed that

650

AgNPs were uniformly introduced into collagen matrix while the existent DXG can fix collagen via

651

Schiff’s base reaction by the simple one-step solution-immersion approach. The physicochemical

652

properties of collagen were effectively improved on account of DXG crosslinking and AgNPs

653

incorporation. As expected, collagen/DXG-AgNPs composite dressing exhibited robust antibacterial

654

activity against Gram-positive and Gram-negative bacteria. In addition, the collagen/DXG-AgNPs

655

composite

656

Collagen/DXG-AgNPs composite dressings could accelerate the deposition of collagen and

657

therefore effectively promote full-thickness burns healing without scar formation. Overall, the

658

collagen/DXG-AgNPs composite dressings have prospective applications in biomaterials as

659

antibacterial wound dressing, hemostatic or blood absorption sponge.

dressing

had

good

blood

compatibility

and

well

cytocompatibility.

660 661

ASSOCIATED CONTENT

662

Supporting Information

663

Additional table and figures (such as AFM, SEM, and optical microscope photos of L929 cells).

664 665

AUTHOR INFORMATION

666

Corresponding Authors

667

*E-mail: [email protected] (D. Li).

668

Notes

669

The authors declare no competing financial interest. 33



670 671

ACKNOWLEDGEMENTS

672

This work was financially supported by the Key Research and Development Project of Sichuan

673

Province (SCST18ZDYF1426), Project of Youth Science and Technology Innovation Research

674

Team of Sichuan Province (2017TD0010) and the Fundamental Research Funds for the Central

675

Universities (2012017yjsy173).

676 677

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41



Table of Content

870

871 872

Synopsis: Antibacterial collagen-based wound dressing was developed by homogeneously

873

introducing greenly synthetic AgNPs and simultaneously crosslinking with dialdehyde xanthan

874

gum.

42


Page 42 of 42

Fabrication of Antibacterial Collagen-Based Composite Wound

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