Curcumin incorporation into an oxidized cellulose nanofiber-polyvinyl alcohol hydrogel system promotes wound healing

Journal Pre-proof Curcumin incorporation into an oxidized cellulose nanofiberpolyvinyl alcohol hydrogel system promotes wound healing

Anha Afrin Shefa, Tamanna Sultana, Myeong Ki Park, Sun Young Lee, Jae-Gyoung Gwon, Byong-Taek Lee PII:

S0264-1275(19)30751-8

DOI:

https://doi.org/10.1016/j.matdes.2019.108313

Reference:

JMADE 108313

To appear in:

Materials & Design

Received date:

26 August 2019

Revised date:

25 October 2019

Accepted date:

26 October 2019

Please cite this article as: A.A. Shefa, T. Sultana, M.K. Park, et al., Curcumin incorporation into an oxidized cellulose nanofiber-polyvinyl alcohol hydrogel system promotes wound healing, Materials & Design(2019), https://doi.org/10.1016/ j.matdes.2019.108313

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© 2019 Published by Elsevier.

Journal Pre-proof Curcumin Incorporation into an Oxidized Cellulose Nanofiber-Polyvinyl Alcohol Hydrogel System Promotes Wound Healing Anha Afrin Shefaa, Tamanna Sultana a, Myeong Ki Parkb, Sun Young Leec, Jae-Gyoung Gwon c, Byong-Taek Leeab a

Department of Regenerative Medicine, College of Medicine, Soonchunhyang University Cheonan-31151, Republic of Korea

b

Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University 366-1, Cheonan-31151, Republic of Korea

c

Division of Environmental Material Engineering, Department of Forest Products, Korea

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Forest Research Institute, Republic of Korea *Corresponding Author:

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Byong Taek Lee

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E-mail: [email protected] Phone: +82-41-570-2427,

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Conflict of interest: The authors declare no conflict of interest. Abstract:

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Incorporation of curcumin (Cur) into a hydrogel system is an interesting approach to treat fullthickness skin wounds because Cur can potentiate healing by affecting different stages of the

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wound healing. Due to its hydrophobicity, Cur solubilization is a great challange. Thus, Cur solubilization by pluronic F-127, gelation capacity of polyvinyl alcohol (PVA) and porosity

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enhancement by TEMPO-oxidized cellulose nanofiber (TOCN) can inaugurate a biocompatible and biodegradable hydrogel system for Cur delivery. In this study, a Cur incorporated physically crosslinked TOCN-PVA-Cur hydrogel was prepared by a freeze-thaw process, which released Cur to accelerate wound healing. The viscosity of the hydrogel was increased by increasing the PVA concentration. In vitro studies revealed that, L929 fibroblast cells internalized curcumin within 4 h of incubation. After the application of the TOCN-PVACur into rat full-thickness skin wounds, the percentage of wound closures was increased compared to that in the control group. Distinct neo-epidermise and granulation tissue formed in hydrogel treated groups and collagen fibers accumulated near defect areas at the two weeks after treatment. These results showed show that the delivery of curcumin by TOCN-PVA-Cur hydrogel can be an effective method for promoting natural wound healing processes. Keywords: wound healing; hydrogel; curcumin; oxidized cellulose nanofiber; polyvinyl alcohol. 1. Introduction

Journal Pre-proof Traumatic skin loss due to surgeries, burns, and accidents followed by delayed wound healing is one of the most psychologically and physically debilitating injuries. There are approximately 100 million traumatic and 300 million chronic wound patients around the world [1, 2]. The prevalence of chronic wounds occurs as a result of microbial infections [3], peripheral vascular disease, type 2 diabetes and metabolic syndrome [1, 4, 5]. Therefore wound site coverage with a suitable dressing is a fundamental need, not only to protect the wound from external risks [6, 7] but also to accelerate wound healing by stimulating cell proliferation and migration factors [8-10]. Moreover, full-thickness wound closure by natural wound contraction force is a slow process [11, 12] and skin grafting is fast and effective in

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closing a defect [13]. In contrast, skin grafting creates a secondary wound which causes discomfort and poses cosmetic issues [14, 15]. Injectable hydrogel therapies can lead to

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efficient wound contraction and eliminate surgical procedure related secondary defect and

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scar [16, 17]. Curently, a number of hydrogels are being widely studied as wound dressings [18-20]; however most of them were proved to be inefficient for long-term patient care,

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particularly for those with large burn areas, infected and severe chronic wounds [19, 21]. Thus, a multi-functional injectable wound healing hydrogel has been proposed for skin defect

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therapies with an emphasis on anti-inflammatory, protective properties and can be shaped to the wound bed that ultimately aid wound healing.

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Although, hydrogels can be prepared by both physical and chemical crosslinking, the need for physically crosslinked gels has increased [22] in order to avoid toxicity related to chemical

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crosslinking agents. Crosslinking from physical stimuli (such as: temperature, pressure, light, electric fields) is therefore preferred for hydrogel preparation [23]. Although, thermosensitive hydrogels are gaining popularity due to their convenience of application at various sites of action [24], their low mechanical strength and slow temperature responses have motivated researchers to overcome drawbacks of thermosensitive and other stimuli sensitive hydrogels. Preformed hydrogel prepared from freeze–thawing cycle can overcome several weaknesses of thermo-responsive and other hydrogels, as preformed hydrogel are insensitive to residual materials and thermostable (in contrast to thermo-responsive hydrogel respectively) [25]. Curcumin (1,7‐ Bis (4‐ hydroxy‐ 3‐ methoxyphenyl) ‐ 1,6‐ heptadiene‐ 3,5‐ dione from Curcuma longa has classically been used as a herbal medicine due to its antimicrobial, antiinflammatory and antioxidant functions [26, 27]. The efficacy of curcumin (Cur) as a wound healing agent relies on its antioxidant properties and in particular, its ability to eliminate ROS (Reactive Oxygen Species) and LPx (Lipid Peroxidation) at the wound area [28-30]. Moreover, curcumin increases cellular proliferation, collagen synthesis, collagen maturation, 2

Journal Pre-proof cross linking of collagen, [30] as well as biosynthesis of extracellular matrix (ECM) [31, 32] at the wound site. However, hydrophobicity of curcumin makes it difficult to be incorporated into hydrogel system, as direct dispersion of curcumin into water is impossible. Thus, a polymer (Pluronic F-127) with both hydrophilicity and hydrophobicity has been suggested as possible solution to disperse curcumin in water [33], since use of large amount of organic solvents (acetone, ethanol, dimethyl sulfoxide and dimethyl formamide) could reduce biocompatibility of hydrogels [34]. Repetitive freeze–thawing cycle of PVA (Poly-vinyl alcohol) solution (with high molecular weight and high degree of hydrolysis 99%) can result in a hydrogel network [35, 36]. Moreover, this type of preformed hydrogel can have its

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characteristics tuned by adjusting both PVA concentration and freeze-thawing cycles. (PVA) based hydrogels are non-toxic, non-carcinogenic and mechanically strong, with superior

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swelling ratio and elastic nature, [36-38]. However, they lack cell adhesion phenomena on

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PVA hydrogel surfaces [39]. They also lack of interconnected porosity, which is often a problem to deal with [39, 40]. So, TEMPO(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl)-oxidized

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cellulose nanofiber (TOCN) has been introduced to the PVA hydrogel system to increase biocompatibility [41, 42], and to provide a stable 3D environment for cell growth [43] and

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improvement of wound healing properties [44]. Moreover, TEMPO oxidation helps cellulose fibrils to be well dispersed in aqueous solution compared to non-oxidized and bacterial

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cellulose [45-47].

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Although, a number of hydrogels containing the above mentioned components are available, to the best of our knowledge, curcumin eluting TOCN-PVA hydrogel has not been studied yet to address issues related to curcumin loading in hydrogel system and faster wound healing. We propose a multi-functional wound-healing hydrogel TOCN-PVA-Cur to overcome drawbacks of currently available hydrogels. In the current study, we used freeze-thaw process to prepare curcumin-eluting preformed TOCN-PVA hydrogel to facilitate full thickness skin wound healing at the earliest possible time. We propose that TOCN-PVA-Cur is easy to apply regardless of wound size, biocompatible and effective as a wound dressing material based on in vitro and in vivo experiments. 2

Experimental Section

2.1 Materials: TEMPO(2,2,6,6-Tetramethylpiperidin-1-yl)oxyl)-oxidized cellulose nanofiber (TOCN) (conc. 1%) was kindly provided by Korea Forest Research Institute, South Korea, and was prepared by a previously described method [42, 45, 46, 48] and used without further modification. In 3

Journal Pre-proof brief, a high speed homogenizer (M-110EH30, Microfluidics, USA) was used to disperse cellulose (1.95 g, 12 mmol anhydroglucose units) in distilled water. After that, the oxidizing solution containing TEMPO (30 g, 0.19 mmol, Sigma Aldrich, USA), NaBr (0.63 g, 6.1 mmol; Sigma Aldrich, USA) and NaOCl (1.76 M solution, 15 ml, 2.64 mmol; Sigma Aldrich, USA) was prepared, then added to cellulose solution and mixed well. Then pH 10 was maintained by adding 0.5 M NaOH solution until the reaction came into an end. To destroy activity of residual NaOCl, 5 ml methanol was added to the reaction mixture and 0.5M HCl was added to maintain pH 7. Finally, the resulting mixture was centrifused to get water soluble oxidized cellulose drivative and pellets of surface carboxylated cellulose nanocrystals.

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To purify TOCN, centrifugation and re-dispersion of cellulose derivative was repeated, followed by dialysis against distilled water. The degree of oxidation was reported to be

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ranging from 55 to 90%. Curcumin powder (Mw 368.38), polyvinyl alcohol (Mw 89,000-

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98,000, 99% hydrolyzed) and Pluronic F-127 (non-ionic powder) were purchased from Sigma Aldrich (USA). The mouse L929 cell line was provided by Korea Cell Line Bank.

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2.2 Preparation of hydrogel:

Due to hydrophobic characteristics of curcumin, incorporation of curcumin with hydrogel

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system is a great challenge. To solve this problem, ethanol (100%) and pluronic® (4%) were used to solubilize curcumin (Cur). Cur (0.3 g) was dissolved in ethanol (1ml, 100%) and

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aqueous pluronic® (9 ml, 4%) solution was mixed, resulting in Cur/Plu solution. Subsequently, 20ml water was added to the Cur-Plu solution. Different amounts of PVA

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powder (0.5, 0.75 and 1 g) were added to each of 10ml Cur/Plu solution and heated (50-70oC) to make 5PVA-Cur/Plu, 7.5PVA-Cur/Plu and 10PVA-Cur/Plu samples respectively. To each PVA-Cur/Plu solution, 10ml TOCN was added and mixed well by constant stirring. Hydrogels were formed from by freezing and thawing. Each cycle of freeze-thawing involved lowering the temperature to −20 °C for 12 h and thawing for 1h at 37 °C. The hydrogels were stored at 4 °C until further use. Except SEM observation (freeze dried), in all of the experiments, this freeze-thaw hydrogels were used. Sample Name

(TOCN:PVA:Cur/Plu) Weight Ratio (g/ml)

(TOCN:PVA:Cur/Plu) Percent Ration (%)

TOCN-5PVA-Cur

0.01:0.05:0.0074:0.005

1:5:0.74:0.5

TOCN-7.5PVA-Cur

0.01:0.075:0.0074:0.005

1:7.5:0.74:0.5

TOCN-10PVA-Cur 0.01:0.1:0.0074:0.005 Table 1: Composition of wound healing hydrogel.

1:10:0.74:0.5

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Scheme 1. Schematic overview of fabrication of TOCN-PVA-Cur hydrogel and probable chemical mechanism.

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2.3 Morphology Analysis:

Morphology of freeze dried (frozen at -80oC and freeze dried at -80oC) hydrogel cross-

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sections was observed via scanning electron microscopy (SEM, JEOL, JSM-6701F, Tokyo, Japan) equipped with energy-dispersive spectroscopy (EDS). Freeze dried hydrogels were cut

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into small pieces and cross-sections were placed on the SEM sample holder and coated with a platinum sputter coater. 10 kV acceleration voltage was used to obtain SEM images. Wet samples were observed at 488 nm excitation by a confocal fluorescent microscope (Olympus, FV10i-W, Tokyo, Japan), and FV10i-ASW 2.0 Viewer software were used to visualize and analyze images.

2.4 In vitro degradation behavior: The in vitro hydrolytic degradation of hydrogels was conducted in PBS (pH 7.4) at 37 ºC. Briefly, hydrogels of known dry weights (Wi) were incubated in PBS under gentle mechanical agitation for the duration of the study (1, 2, 4, 6, 8, 10, 12, 15, 21 Days). The PBS was refreshed daily. After specific time intervals PBS was removed from the samples, rinsed with distilled water and final weight (Wf) was measured. For statistical evaluation, triplicates of each sample groups were experimented. The percentage of weight loss was determined by the following equation (2) 𝑷𝒆𝒓𝒄𝒆𝒏𝒕 𝒘𝒆𝒊𝒈𝒉𝒕 𝒍𝒐𝒔𝒔 =

𝑾𝒊 −𝑾𝒇 𝑾𝒊

× 𝟏𝟎𝟎

(2) 5

Journal Pre-proof 2.5 Viscosity Analysis: Viscosities

of

hydrogels

after

each

freeze-thaw

cycle

were

measured

by

a viscometer (Brookfield DV2TRV Viscometer-Standard Viscosity). Viscosities at 37 oC temperature by using rotor (SC4-21) at the range of 1-100 rpm. For statistical evaluation, triplicates of each sample groups were experimented. 2.6 Cells viability assay: Cell viability was determined by colorimetric assay utilizing MTT (Gibco™3-[4,5dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide; Thermo Fisher Scientific, USA). L929 cells were cultured in Gibco® RPMI (Roswell Park Memorial Institute)- 1640 medium

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(Thermo Fisher Scientific, USA), 1% Penicillin(100 U/mL) streptomycin (100 U/mL) antibiotic (PS; ThermoFisher Scientific, USA) and 10% fetal bovine serum (FBS; Thermo

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Fisher Scientific, USA). To check effect of TOCN, PVA and Cur on cell viability only

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5%PVA, TOCN-5PVA and TOCN-5PVA and TOCN5PVA-Cur hydrogels were used. Moreover, 1, 3, 7 day cell viability of curcumin loaded hydrogels (TOCN-5PVA-Cur, TOCN-

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7.5PVA-Cur and TOCN-10PVA-Cur) were checked. In brief, three sets of samples were subjected to sterilization by UV-irradiation and incubated with RPMI growth medium for 3

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days. L929 cells were seeded in 24-well plate (1x104 per well) and incubated until cells were confluent. Then, cells were incubated with sample extracted media at specific time interval.

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After specific time period, 100 µl MTT solutions (5 mg/mL) was added to each well and incubated in a CO2 incubator (5% CO2, 37 oC). The solution was carefully removed, followed

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by addition of 1000 µL dimethyl sulfoxide (DMSO 99.5%; Daejung Chemicals & Metals CO., Korea) each well to solubilize MTT. The resulted solutions were measured in triplicates at 595 nm using an ELISA reader (Infinite® F50,Tecan Trading AG, Switzerland) and cell viability was measured by the following equation (3) 𝐶𝑒𝑙𝑙 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =

𝐴𝑏𝑠𝑟𝑜𝑏𝑎𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑒𝑙𝑢𝑡𝑒𝑑 𝑚𝑒𝑑𝑖𝑎 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑜𝑛𝑙𝑦 𝑚𝑒𝑑𝑖𝑎 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠

× 100

(3)

2.7 In vitro cell proliferation study: Three sets of samples were subjected to sterilization by UV-irradiation and incubated with RPMI growth medium for 3 days (Curcumin concentration 1100-1200µg/ml). L929 cells were seeded in confocal dish (SPL Life Sciences Co., Ltd. South Korea) (1x104 cells per well) as different experimental groups and incubated in 37°C incubator at 5% CO2 for 1, 3 and 7 days. At specific time interval, cells were stained. Briefly, fixation (4% paraformaldehyde; Sigma-Aldrich), and staining were conducted sequentially with respective reagents. Nuclei and cell cytoskeleton were stained with HOECHST-33342 (2'-[4-ethoxyphenyl]-5-[4-methyl1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate; 1 μg/mL; Invitrogen, 6

Journal Pre-proof USA) and

fluorescein isothiocyanate (FIITC)-conjugated phalloidin (25 μg/mL; Sigma-

Aldrich, USA) respectively. Finally, a Laser Scanning Confocal fluorescent microscope and FV10i-ASW 2.0 Viewer software were used to visualize and analyze images. Number of cells per unit area was quanitified. 2.8 In vitro curcumin release: Curcumin release from three types of curcumin loaded hydrogels (TOCN-5PVA-Cur, TOCN7.5PVA-Cur, TOCN-10PVA-Cur) was studied in PBS (pH 7.4) for 15 days at 37 °C. Briefly, in a 12-well plate, 1mL hydrogels were placed and incurbated with 1mL PBS. At specific time intervals (1, 2, 3, 4, 5 h and 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 15days) PBS were collected

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from hydrogel and assayed by Biodrop#1005363 Spectrophotomer (Denville Scientific Inc, USA). Cumulative amounts of curcumin were then determined using a standard calibration

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curve. Control samples (hydrogel without curcumin) were also subject to spectrophotometry

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to deduce the interference of components other than curcumin.

UV-vis spectra of pluronic solution, Cur/Plu miscelie solution and three types of sample

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eluted solutions were generated using a Biodrop#1005363 Spectrophotomer (Denville Scientific Inc, USA), to determine the presence of pluronic and Cur/Plu miscelie in the eluted

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solution. To analyze FT-IR (Fourier Transform Infrared Spectroscopy) spectrum of pluronic, curcumin and curcumin/pluronic miscelie Nicolet spectrometer (Nicolet Ia10, Thermo

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Scientific) and OMNIC version 7.3 software were used in the wavelength range of 500–4000 cm−1 at a resolution of about 8 cm−1.

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2.9 In vitro curcumin uptake:

Cellular uptake tests of curcumin from hydrogels were performed in L929 cells. 1×104 cells were seeded on confocal dish (SPL Life Sciences Co., Ltd. South Korea) and grown in RPMI growth media for 24 hours in 37 °C incurbator with 5% CO2 until confluence. Curcumin loaded hydrogels (TOCN-5PVA-Cur, TOCN-7.5PVA-Cur, TOCN-10PVA-Cur) were sterilized by UV-irradiation, applied on predesigned confocal dishes and incubated for 4, 12 and 24 h. During study period, hydrogels were kept floating to release curcumin miceli. After specific time interval, the hydrogel samples and the growth media were removed and cells were washed with PBS and stained with 100 µL of 1 μg/mL HOECHST 33342 (2′-[4ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole

tri-hydrochloride

tri-

hydrate; Invitrogen, USA) for 10 min. Then cells were visualized by confocal fluorescent microscope (Olympus, FV10i-W, Tokyo, Japan), and images were analyzed by FV10i-ASW 2.0 Viewer software. 2.10

Wound contraction and histological analysis of skin defect site: 7

Journal Pre-proof Forty healthy adult male Sprague Dawley male rats (rattus norvegicus) (mean weight 200 g) were purchased for this study, placed in standard cages and food/ water provided. The experimental procedure was approved (SCH19-0020) by Animal Ethics Committee of Soonchunhyang University, South Korea. The rats were housed and adapted to their environment for 7 days before in vivo study. Animals were randomly divided into four groups, named; Control, TOCN-5PVA, TOCN-5PVA-Cur, TOCN-7.5PVA-Cur. At 1st and 2nd week time-point animals were sacrificed. There were 20 animals per time point (4 groups of 5 rats each x 2). On the day of wounding, the rats were anaesthetized with Isofluran (Piramal Critical Care, Schelden Circle, PA). Site to be excised were swept with povidone iodine

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solution. Full thickness 10mm diameter excisional wound was created at a point 7 ± 1 cm from the rat’s ears and tail [49] using a biopsy punch (Kai Industries Co. Ltd, Japan) and

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forceps on the dorsum of each rat and treated with specific type of hydrogel samples. Each

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types of hyrdogels were prepared by freeze thawing and sterilized by UV and injected on the wound site. The wound site was monitored, photographed and the diameter was measured, to the

wound

(%)

according

to

the

𝐼𝑛𝑡𝑖𝑎𝑙 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑤𝑜𝑢𝑛𝑑−𝑛𝑡ℎ 𝑑𝑎𝑦 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑤𝑜𝑢𝑛𝑑 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑤𝑜𝑢𝑛𝑑

following ×100%

equation

(4): (4)

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𝑊𝑜𝑢𝑛𝑑 𝐶𝑙𝑜𝑠𝑢𝑟𝑒 (%) =

closure

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quantify

Skin wound sites were extracted at specific time point (1st week and 2nd week). For this

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purpose, rats were euthanized using an over dose of diethyl ether (Daejung, South Korea). A full-thickness skin sample was extracted from the wound site. Tissue samples were immediately fixed in 10% formaldehyde (Duksan Pure Chemicals, Korea) solution for 48 h at

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room temperature. Tissue samples taken from each of the animals were stored in -80 °C until used for western blot protein analysis. For histological analysis, formaldehyde fixed samples were subject to dehydration and paraffin embedding using previously described method [44]. Paraffin embedded skin samples were cut into sections (7 µm thickness) using a microtome (Thermo Scientific, USA), deparaffinized, and stained by hematoxylin and eosin (H & E), Masson’s trichrome and Picroosirius Red staining. Stained tissue sections were viewed with a BX53 light microscope (Olympus) and photographed with an Olympus DP72 camera. Images were analyzed using Cellsens Software. 2.11

Sample preparation and western blot analysis of skin defect site:

Samples were homogenized and total proteins were extracted using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) and centrifuged. 40 μg of total protein from each sample was electrophoresed in 8% SDSpolyacrylamide gels and blotted onto polyvinylidene difluoride membranes(Bio-Rad, CA, USA). 8

Journal Pre-proof Afterwards, membranes were blocked with 3% BSA (Rocky mountain biologicals, UT, USA) in TBS buffer (400 mM Tris-HCl pH 7.4 and 3 M NaCl. BioSesang, Republic of Korea) containing 0.05% Tween 20 (BioSesang, Republic of Korea), and incubated overnight at 4 °C with the following primary antibodies, including anti-alpha smooth muscle actin (1:3000; Abcam, UK), anti- Fibronectin (1:3000; Abcam, UK), anti- Collagen1 (1:1000; Santa Cruz Biotechnology, TX, USA), anti- e-Cadherin (1:1000; Santa Cruz Biotechnology, TX, USA), and anti-β-actin (1:3000; Santa Cruz Biotechnology, TX, USA) in 3% BSA containing TBST. The blot was washed 3 times with TBST for 5 min and incubated with horseradish peroxidaseconjugated goat anti-rabbit (1:3000; cell signaling, MA, USA) and horse anti-mouse (1:3000;

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cell signaling MA, USA) IgG in 3% BSA contained TBST at room temperature for 1 h. The bound antibody was detected with chemiluminescence reagent (GE health care, Austria) and

Statistical Analysis:

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2.12

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then developed on film.

Statistical analysis was conducted using one-way analysis of variance (one-way ANOVA).

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Results are expressed as mean ± standard deviation.

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3. Results and discussions:

3.1 Preparation of TOCN-PVA-Cur hydrogels: Curcumin has low water solubility and is difficult to dissolve into hydrogel systems, therefore

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a polymer (Pluronic F127) was used to give curcumin a platform for dispersion into the hydrogel [33]. Pluronic is a block co-polymer which consists of hydrophilic ethylene oxide

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(EO) and hydrophobic propylene oxide (PO) blocks arranged in a triblock (Scheme 1). This arrangement results in amphiphilic copolymers, which serve as a cargo space for the adsorption of hydrophobic curcumin. Meanwhile the hydrophilic corona markedly facilitates the dispersion of curcumin into hydrophilic polymer systems like TOCN-PVA via hydrogen bonding [50]. From the previous studies, freeze-thaw mediated gelation and crosslingking mechanism of oxidized cellulose and PVA hydrogels are obvious. In brief, the PVA solution was prepared by dissolving in water and then mixed with TOCN suspension which looks apparently homogeneous at room temperature. In the aqueous environment, PVA molecular chains are mobile and come in intimate contact with each other for a very short time. Carboxyl (– COOH) and hydroxyl (-OH) groups from TOCN, form hydrogen bonds with –OH groups of PVA [51, 52]. So, TOCN also migrates with the PVA chain in the aqueous environment as a result of hydrogen bonding interactions. Upon freezing the molecular motion of the TOCNPVA mixture tends to be restricted to some extent. As the water freezes, TOCN-PVA is 9

Journal Pre-proof expelled toward a polymer rich zone [53, 54], which allows TOCN-PVA chains to come in contact with each other. The intermolecular interaction among the PVA chains probably leads to hydrogen bonding and thus promotes the formation of physically entangled zones or crystallite formation[55, 56]. Then the resulting crystallites act as crosslinks, which produces a three-dimensional network structure (shown in Scheme 1). Such a crosslinked structure withstands higher temperatures. So, the higher the PVA concentration in the aqueous solution, the shorter the distances between entangled polymer zones leading to a larger degree of crosslinking [57, 58], which in turn affects hydrogel viscosity, degradation and curcumin release.

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3.2 Morphology Analysis:

SEM micrograph (Figure 1A) revealed that, cross sections of hydrogel has interconnected

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macro and microspores. PVA hydrogel prepared from freeze-thaw process, lacks porosity

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(Supplimentary Data 1C), but with the addition of TOCN, porosity of the hydrogel increased in TOCN-5PVA-0 hydrogel. With the increase of PVA contents in hydrogels pore size

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decreases (Figure 1A). Gelation of PVA solution due to freeze-thawing results from phase separation into a water-rich phase and a PVA-rich phase [1, 53, 55, 59]. This water rich phase

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resulted in pore formation when subject to drying for SEM observation. As curcumin is a fluorescent component (350-600nm) [60] of the hydrogel system, so

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curcumin was detected by confocal microscopy. The presence of green fluorescence in the hydrogel system supports the presence of curcumin in each of the TOCN-PVA-Cur hydrogels.

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Pores within hydrogel systems are highly desirable for wound healing systems [44] as they aid the transportation of liquids, gas and nutrients [42] and contribute to the swelling capacity and/or density [41] of hydrogel systems. Moreover, highly porous hydrogel system fascilitates difusion of water from and into the hydrogel resulting in polymers and drug release [61]. 3.3 In vitro degradation behavior:

Results of hydrolytic degradation of TOCN-PVA-Cur hydrogels are shown in Figure 1(B). Supplimentary Data: S2(B) showed that, the higher concentration of PVA hydrogel subject to lower degradation and weight loss. But the present study is focused on the curcumin incorporation and its effect on hydrogel system. The degradation of hydrogel is presented as, weight remaining after PBS immersion. Degradation was highest (9% weight remain) in TOCN-5PVA-Cur group (low PVA content) whereas, the lowest degradation (weight remain 41.87%) observed in TOCN-10PVA-0 (high PVA content) after 15 days incubation (Figure 1B). Moreover, Cur incorporated TOCN-PVA hydrogels (TOCN-5PVA-Cur, TOCN7.5PVA-Cur and TOCN-10PVA-CUR) showed higher degradation compared with only 10

Journal Pre-proof TOCN-PVA hydrogels (TOCN-5PVA-0, TOCN-7.5PVA-0, TOCN-10PVA-0) (Figure S2C). Weight loss occurs in TOCN-PVA-Cur hydrogel due to the release of Cur and PVA. So, the overall hydrolytic degradation of PVA hydrogel is dependent on PVA concentration (Supplimentary Data 2B) and Cur release [62], because, in each of the hydrogel systems, TOCN concentration and the curcumin loading concentration is same. Moreover, Cur is incorporated into TOCN-PVA hydrogel system with the help of pluronic triblock system. When comes in contact with fluid, Cur is released from the hydrogel system along with Cur/Plu cargo/misceli. So, the mechanism behind degradation of TOCN-PVA-Cur hydrogel is the release of Cur

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from the hydrogel matrix and dissolution of PVA chain. The initial dissolution of PVA hydrogel results from the release of PVA chains that did not participate in the crystallite

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region formation process and therefore were not incorporated into the physical structure of the

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hydrogel [59]. TOCN-5PVA-Cur hydrogel degraded by 80% within 14 day of incubation in PBS solution releasing PVA and curcumin into the media. So, it is expected that, hydrogel

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degradation will provide sufficient amounts of curcumin to be therapeutically useful in

3.4 Viscosity Analysis:

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healing wounded skin.

The viscosity of the formulation affects injectability of the hydrogele system[63, 64]. Freeze–

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thawing cycles influence the physical properties of PVA hydrogels [65]. The effects of the shear rate (s-1) on the viscosity of the PVA hydrogel system are shown in Figure 1(C). From Figure 1(C),

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it is obvious that, with an increase of PVA concentration from 5% (TOCN-5PVA-Cur) to 10% (TOCN-10PVA-Cur), the shear viscosity range increased significantly (P<0.01) from 44.09-100 Pa.s to -148.0-433.3 Pa.s respectively. By increasing PVA content viscosity can be increased and increase in the shear rate (s-1) can contribute to the decrease of viscosity of the hydrogel (Figure 1C) which suggests the shear-thinning property of hydrogel. Moreover, addition of TOCN results in a marked decrease in viscosity of PVA hydrogel (Figure S2D). Moreover, incorporation of Cur into TOCN-PVA hydrogel system, the viscosity decreased very in-significantly, as quantity of Cur/Pluronic is very low in the final hydrogel formulation. So, TOCN-5PVA and 0-5PVA-0 hydrogel had the lowest viscosities after freeze thaw (Supplimentary data S2). As, TOCN-PVA and PVA hydrogels have a high viscosity at low shear rates (below 50 s−1) they behave as pseudoplastic fluids (non-Newtonian, shear-thinning fluid) [65]. Moreover, PVA concentration strongly affects viscosity of the PVA hydrogel due to the variation of crystallite and hydrogen bonding interactions among PVA chains [57]. The viscosity of the TOCN-7.5PVA hydrogel decreases more rapidly with shear rate compared to 11

Journal Pre-proof hydrogel TOCN-10PVA. Therefore, shear-thinning property of preformed hydrogel enables it to be delivered at application site by injection with minimum damage to the hydrogel dispenser. For, ease of application, TOCN-5PVA, TOCN-7.5PVA (shear viscosity range 44.09100 Pa.s) hydrogels prepared from freeze thaw cycle 1 were used for further in vivo

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experiments.

Figure 1: SEM micrograph and confocal microscopic fluorescence images (A), in vitro degradation behavior (B) and viscosity (C) of different kinds of hydrogel samples. Curcumin incorporation provided fluorescence property to the TOCN-PVA hydrogel. Weight loss due to hydrolytic degradation and viscosity property of hydrogel is dependent on PVA concentration. Three independent experiments were conducted for degradation and viscosity analysis. The viscosity of TOCN-10PVA-Cur is significantly (One way ANOVA; P<0.01) higher than TOCN-7.5PVA-Cur and TOCN-5PVA-Cur. 3.5 Cell viability assay: Viability of L929 cells was determined by utilizing MTT assay. Figure 2(A) suggests that, approximately 100% cell viability was obtained by 25% hydrogel extracted media. More than 70% of cells were viable in 100% hydrogel extracted media. According to curcumin release study, it is expected that, 3days incubation of hydrogels in media would release 1258.05, 12

Journal Pre-proof 957.9 and 951 µg/mL curcumin from TOCN-5PVA-Cur, TOCN-7.5PVA-Cur and TOCN10PVA-Cur samples respectively. Even-though the sample eluted curcumin concentration was theoretically high enough to cause cell apoptosis [66], in our study it was found that L929 fibroblast cell viability remained at 70% or more to be used as wound healing agent. To confirm percent cell viability of hydrogels (TOCN-5PVA-Cur, TOCN-7.5PVA-Cur and TOCN-10PVA-Cur) at different time point, cells were incubated with hydrogel extracted media, which showed that, even after 7day all of the hydrogel groups showed similar pattern of cell viability

84.12±0.7% (TOCN-5PVA-Cur), 83.3±0.2% (TOCN-7.5PVA-Cur) and

85.51±1.6% (TOCN-10PVA-Cur) compared to control. There was no statistically significant

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difference among hydrogel groups.

The results of cell proliferation study in L929 cells in hydrogel extracted media presented in

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Figure 2C. Cells incubated in sample extracted media showed increased cell proliferation with

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the span of time (from day 1 to 7) (Figure 2C&D). The differences among sample groups were insignificant. So, it can be ascertaine that, curcumin released from hydrogel will not

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hamper growth of cultured fibroblast cells when compared with hydrogel without curcumin (TOCN-5PVA-0). Biocompatibility and desired biological activity is the fundamental criteria

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of a polymer system to be used for biomedical applications [43].

Figure 2: Effect of TOCN and Cur incorporation in cell viability of hydrogel (A); cell viability of Cur incorporated hydrogel extracted media at different time point (1, 3 and 7days) 13

Journal Pre-proof (B); Effect of curcumin hydrogel extracted media on L929 cell after incubation for 1, 3 and 7days(C). F-actin and nuclei visualized by FITC and HOECHST respectively. There is no

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statistical significant difference among hydrogel groups.

Figure 3: Quantitative curcumin release from hydrogel into PBS (A); Cellular uptake of

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curcumin from hydrogel systems at 4-24 h in L929 cells (B); UV-vis spectra of curcumin released from hydrogel (C); Cellular uptake mechanism of curcumin (D). Curcumin release is sustained throughout 15 days and within 4 h curcumin is uptaken by L929 cells. For curcumin release study, three sets of samples were examined and no statistical significance was found between TOCN-5PVA-Cur and TOCN-10PVA-Cur hydrogel groups. But after 1day incubation, TOCN-5PVA-Cur groups released significantly (P<0.05; One way ANOVA) higher amount of curcumin compared to TOCN-7.5PVA-Cur and TOCN-10PVA-Cur. 3.6 In Vitro Curcumin release: Figure 3(A) shows curmulative release profiles of curcumin from different hydrogel samples in PBS (pH 7.4). Curcumin release was highest in TOCN-5PVA-Cur sample. The hydrogels containing 7.5% and 10% PVA released lower amounts of curcumin. In all of the hydrogels, curcumin release increased dramatically after 1day of incurbation. Curcumin loading concentration was 0.0074 g/ml of hydrogel. At 1h of incurbation of TOCN-5PVA-Cur and TOCN-7.5PVA-Cur samples, the amount of curcumin released was 75.66 µg/mL and 62.33 14

Journal Pre-proof µg/mL respectively. On the other hand, TOCN-10PVA-Cur hydrogel released only 8.67 µg/mL in hydrogel eluted PBS. Over subsequent hours, curcumin release was highest from TOCN-5PVA-Cur hydrogel. Following 3days incubation of hydrogels in media, 1258.1, 957.9 and 951 µg/mL curcumin was released from TOCN-5PVA-Cur, TOCn7.5PVA-Cur and TOCN-10PVA-Cur samples respectively. The lower amount of curcumin release from the TOCN-10PVA-Cur hydrogel is attributed to the decreased hydrolysis phenomena of the PVA rich hydrogel systems. Higher amount of PVA results in stronger hydrogen bonding interactions among the TOCN-7.5PVA-Cur and TOCN-10PVA-Cur chains, so the Cur/Plu molecurles are trapped within hydrogel networks. On the other hand, weak hydrophilic

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interactions among TOCN-5PVA chains results in the release of Cur/Plu molecurles, which also has hydrophilic characteristics [67]. Moreover, approximately 37.9597±0.02%,

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28.8864±0.01% and 26.0148±0.07 % of loaded drug was released from TOCN-5PVA-Cur,

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TOCN-7.5PVA and TOCN-10PVA-Cur hydrogels respetively (supplimentary data 2C). Figure 3(C) showed that, sample incubated in PBS for 4hours releases Cur/Plu miscelie. Red

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dotted region showed the presence of Cur/Plu miscelie and black dotted region showed the Pluronic intensity region [68]. A broad peak observed around 350-475 nm for Cur/Plu

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solution, where, the intensity of Cur/Plu released from hydrogels are lower. Intensity peak of TOCN-10PVA-Cur sample released solution has lowest intensity while compared with

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TOCN-5PVA-Cur released. This supports that, TOCN-5PVA-Cur sample releases higher amount of curcumin compared to TOCN-7.5PVA-Cur and TOCN-10PVA-Cur.

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3.7 In Vitro curcumin uptake by fibroblast: As a lipophilic molecule, curcumin is expected to be localized in the cellular membrane [69]. To assess uptake behavior, murine fibroblast L929 cells were treated with curcumin hydrogel and UV-Vis spectra of hydrogel eluted media were generated to confirm presence of curcumin in media. Hydrogel eluted curcumin was internalized by L929 cells and at 4 h of incubation, curcumin started to accumulate within the cell membrane (Figure 3B). To gain an idea of the specific time required for cells to uptake curcumin, L929 cells were treated with curcumin hydrogel eluted media for different time periods (4, 12 and 24 h). Fluorescence images were captured by confocal microscope and show emission of fluorescence from the cellular curcumin. Since the majority of the cell volume is occupied by the nucleus with very little cytoplasm, the emission could be from both the cytoplasm and the nucleus. Moreover, the fluorescence from curcumin was green and that from HOECHST as red (Figure 3A). The superimposed images clearly indicate green and red areas overlap as yellow signal, confirming the co-localization of curcumin and HOECHST in the cells. 15

Journal Pre-proof Figure 3B provides evidence that curcumin fluorescence within the cell could be observed as early as 4 h of incubation. Therefore an incubation time of 4h appears to be enough for cellular uptake of curcumin. Another study documented that, 0.5-2 h is enough for maximum cellular uptake of curcumin by cancer cells [69]. Curcumin release (Figure 3A) show that, within 4 h of incubation of hydrogels in PBS, 268(5PVA), 126(7.5PVA) and 114(10PVA) µg/ml curcumin was released. Those curcumin concentrations are enough for cellular uptake. Though it can be inferred from the present study that, curcumin uptake rate is dependent on incubation time, the mechanism of cellular upake by membrane partitioning (concentration gradient) could not be ignored as there are

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other possible interpretations[70]. The cellular internalization mechanism of curcumin in the form of nanoparticles is through an endocytic process [71, 72]. Moreover, Sahay et al.

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elucidated an endocytic mechanism for pluronic block copolymers and clathrin/caveolae-

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mediated mechanism for phagocytosis of pluronic block was documented [73]. The authors described that, pluronic inserts hydrophobic chains into lipid bilayers and decreases

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membrane microviscosity [73]. So, Cur-Plu micelles showed better cellular uptake compared to the free curcumin [66] showed in Figure 2(C).

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3.8 Wound contraction and histological analysis of skin defect site: TOCN and curcumin in combination possess advantageous properties in regards to wound-

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healing, anti-infective and skin regeneration [28, 30]. TOCN is highly biocompatible and has favorable mechanical characteristics, along with a porous structure that mimics native skin

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ECM [44]. Being hydrophilic in nature, TOCN ensures that the wound will retain moisture. The wound healing potential of TOCN-7.5PVA-Cur, TOCN-5PVA-Cur and TOCN-5PVA hydrogel was evaluated by applying them onto full-thickness excision wounds in a rat model and comparing the results to open wound control. Figure 4(A) shows representative photographs of the control group, hydrogel group and the hydrogel with curcumin at 0, 1, and 2 weeks postsurgery. 1 week after hydrogel application, the TOCN-5PVA-Cur and TOCN-7.5PVA-Cur groups evidenced significant wound closure (28.8 ± 1.3 % and 29.9±1.7 % respectively) in comparison to the control (8.3 ± 1.13 %). While the wound size in all groups decreased as time elapsed, the wound size of the curcumin loaded hydrogel (TOCN-5PVA-Cur and TOCN7.5PVA-Cur) groups was smaller than the other two groups, and the wounds had almost closed by 2 weeks. In addition, the hydrogel groups had a superior healing effect compared to control group. Measurement of the wound size confirmed the abovementioned results (Figure 4B). 2week post-surgery, the TOCN-5PVA-Cur (81.3±1.3 %) and TOCN-7.5PVACur (80.3±1.4 %) groups showed significantly better healing effects compared to the control 16

Journal Pre-proof (35.63±1.3 %) and TOCN-5PVA (50.63±1.12 %) groups (Figure 4B). Masson’s trichrome staining of 2weeks post-surgery skin tissue section (Figure 4C) showed that, central to the wound site collagen fiber formation potentiated wound healing. It is noteworthy that, TOCN and PVA being biocompatible and bioactive efficiently facilitate healing; however, the curcumin incorporation heightened the wound healing activity of the TOCN-PVA hydrogel

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system enabling faster wound healing.

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Figure 4. Representative images (scale bar represents 10mm) (A) and percent wound closure (B), The error bars indicate the standard deviation of multiple measurements, ***P < 0.01

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compared to control (One way ANOVA). Five animals were used in each of the groups to conduct wound healing study.

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Dermal regeneration and re-epithelialization of the wounds treated with various hydrogel compositions were examined with H&E (hematoxylin and eosin) staining and presented in Figure 5 (A,B). 2week post-surgery, except control group, the neo-epithelium formed in the wound defects in the TOCN5PVA-Cur and TOCN-7.5PVA-Cur groups (Figure 5B). Thick granulation tissue appeared in all sample groups compared to control. Among the TOCN5PVA-Cur and TOCN-7.5PVA-Cur groups, granulation tissue thickness was higher in TOCN-7.5PVA-Cur group. The total neo-epithelium was thicker in the curcumin loaded hydrogel group than the control group at 2 weeks post-surgery. Wound healing mechanism of hydrogel in different stages shown in Figure 5(C).We postulate that, after application of TOCN-5PVA-Cur or TOCN-7.5PVA-Cur hydrogel at fill-thickness wound site, wound area will be covered inhibitng microbial invasion and reducing inflammation. Then, curcumin will be released from the hydrogel, internalized by cells around the wound area and leading toward wound contraction. Moreover, TOCN and PVA not being capable to dermal absoption, will be discarded along with scar tissue. 17

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Figure 5. H&E staining of skin tissue following hydrogel application after 1(A) and 2 weeks (B). NE=neo-epidermis formation; GT=Granulation Tissue. Black dotted region depicts the remaining wound area, which represents that, wound contraction is highest in TOCN-5PVA and TOCN-7.5PVA-Cur. Wound healing mechanism of hydrogel in different stages (C). Neoepidermis formation was higher in TOCN-5PVA-Cur and TOCN-7.5PVA-Cur hydrogel samples. 3.9 Western blot analysis of skin defect site: Western blot (WB) analysis of α-SMA (smooth muscle actin), fibronectin, collagen-I and Ecadherin protein marker of the regenerated tissue are shown in Figure 6. Quantitative analysis of skin tissue regeneration profiles by western blot analysis revealed both the ECM deposition and maturation of tissue at the wound bed.

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Journal Pre-proof The ECM component, fibronectin facilitates cell reorganization and movement by connecting cells directly with collagen fibers through integrin receptor mechanisms [74]. In the present study, abundant fibronectin was found at 1 week post-surgery (Figure 6A). In the curcumin containing samples, expression of fibronectin protein is higher than that of the control and 5PVA-0 groups. After injury, the plasma form of fibronectin is incorporated into fibrin clots and helps in homeostasis by provisional matrix formation [75]. After 2 weeks of injury, fibronectin expression is lowered. In response to wounding, dermal fibroblasts and keratinocytes [76] produce cellular fibronectin which is assembled into a complex threedimensional fibrillar network on the migrating cell surface [77]. Polarized fibroblasts along

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the fibronectin fibrils facilitate stable collagen I/III fibrillar network formation through cellular integrin receptor mechanisms. Moreover, fibronectin in the wound site is also vital

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for regulating the neovascularization of granulation tissue during the recovery of tissue injury

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and once the wound becomes re-epithelialized, fibronectin progressively disappears [75, 76]. Extensive deposition of collagen-I fibers was observed in the hydrogel treated wound bed

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compared with the control groups at 2 weeks post-surgery (Figure 6). Moreover, there was more collagen accumulation and thick wavy collagen fibers in the curcumin loaded hydrogel

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group (Figure 5C Masson’s trichrome and picrosirius red staining). Quantitative analysis (Figure 6) revealed the collagen content with the curcumin loaded hydrogels group and only

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hydrogel group was significantly higher than the control group at 2 week post-surgery. This evidence suggests improved collagen biosynthetic capacity for TOCN-PVA-Cur hydrogel

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treated skin [30], as curcumin helps with fibronectin fibril formation. Expression of α-SMA is the major identifying feature of the myofibroblast within cytoskeletal stress fibers. With the exception of the control group, α-SMA appeared in wound sites after 7days of the hydrogel treatment (Figure 6C). But, by week 2, all of the skin wounds synthesized α-SMA. However, in full-thickness excisional wounds in rats after 1 week of healing, before myofibroblasts first appear, proto-myofibroblast, initiates initial contraction [78]. Moreover, α-SMA expression is significantly associated with microvascular density and myofibroblasts [79, 80] as well as facilitates the injury repair through growth factor secretion, contractile forces and matrix remodeling. In a nut shell, enhanced deposition of collagen, and fibronectin proteins by curcumin hydrogel treated wounds suggests that curcumin helps in the remodeling of tissues and provides strength to the wounded skin. As a major cell-cell adhesion molecule, e‐ cadherin expression has a close relationship with cell maturation, stratification and wound healing [81]. Together e‐ cadherin and fibronectin play a vital role in epithelial–mesenchymal transition (EMT) 19

Journal Pre-proof during wound healing, embryogenesis and tumorigenesis [82, 83]]. Fibronectin expression diminished after 2 week post-surgery; however, it was most pronounced after 1 week. Moreover, e-cadherin expression is also higher in most of the sample groups and control group at day 7, but e-cadherin expression is almost close to normal skin at day 14th. In particular, after 14th day, TOCN-7.5PVA-Cur group got normal skin like e-cadherin expression level, but even after 14th day control group skin contain high amount of e-cadherin which might hamper wound closure. Classical EMT is characterized by loss of epithelial protein e-cadherin, and an increase in mesenchymal proteins, like, fibronectin [84]. Moreover, wound healing could be slower down due to impairment of fibroblast growth and EMT [85,

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86]. Similar to EMT, migration of epithelial sheets under injury stimulation is a key feature of re-epithelialization [87]. So, it could be speculated that e-cadherin expression is pronounced

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after the 1st week post injury and e-cadherin expression is diminished after the 2 weeks in

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curcumin loaded sample groups due to re-epithelialization and after neo-epidermis (Figure 5B) formation. Moreover, curcumin loading negatively affects e-cadherin expression [88] when

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compared with the control and TOCN-5PVA-0 groups.

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Journal Pre-proof Figure. 6. Representative Protein expression fold change vs normal skin (A), blots of the protein level (B) of α-SMA, fibronectin, collagen-I, e-cadherin derived from western blot analysis of rat skin after 1st (1W) and 2nd (2W) week treatment of hydrogels and histological analysis (PS red and MT staining) sections of skin after 2weeks of rat skin application of hydrogel (scale bar represents 500µm) (C). Results obtained from 3 separate experiments. Where, *P<0.05, **P<0.01 and ***P < 0.001was accepted as statistically significant (One way ANOVA). Here, 5PVA-0=TOCN-5PVA-0, 5PVA-Cur=TOCN-5PVA-Cur, 7.5PVACur=TOCN-5PVA-Cur, 10PVA-Cur=TOCN-10PVA-Cur; PS=Picrosirius; MT= Masson’s Trichrome Conclusion:

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In conclusion, pluronic aided curcumin incorporation into TOCN and PVA polymers and

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subsequent hydrogel preparation was conducted via freeze-thaw process. TOCN-5PVA-Cur

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and TOCN-7.5PVA-Cur showed moderate viscosity and considerable degradation and subsequent curcumin release. Meanwhile, curcumin uptake by L929 cells was facilitated by

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the pluronic polymer system. Notably, wound healing markers were expressed at the wound bed which resulted in significant wound contraction after 2 weeks of curcumin loaded

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hydrogel application in rat skin excisional wound model. Newly developed TOCN-5PVA-Cur and TOCN-7.5PVA-Cur hydrogels proved to be efficient in wound contraction, by enhancing

scar formation.

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the rate of collagen organization without the need for and associated secondary defects and

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Research drawbacks: Though a number of studies have been carried out to evaluate in vitro and in vivo wound healing performance of the preformed TOCN-PVA-Cur hydrogels, due to limited laboratory facilities quantification of PVA, TOCN, pluronic release from the hydrogel system could not be determined. Moreover, wound healing activity could be better explained if diseased model could have been established, which we have taken under consideration for our future research. Acknowledgements: Korea Forest Research Institute (FP0400-2016-01-2016), National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2015R1A6A1A03032522) and partially Soonchunhyang University supported this research work. Data Availability: The raw/processed data required to reproduce these findings cannot be shared at this time due to legal or ethical reasons. References

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Wu, Copper Silicate Hollow MicrospheresIncorporated Scaffolds for Chemo-Photothermal Therapy of Melanoma and Tissue Healing, ACS nano 12(3) (2018) 2695-2707. [5] K.K. Chereddy, A. Lopes, S. Koussoroplis, V. Payen, C. Moia, H. Zhu, P. Sonveaux, P. Carmeliet, A. des Rieux, G. Vandermeulen, V. Préat, Combined effects of PLGA and vascular endothelial growth factor promote the healing of non-diabetic and diabetic wounds, Nanomedicine 11(8) (2015) 1975-1984. [6] Y. Zhou, L. Gao, J. Peng, M. Xing, Y. Han, X. Wang, Y. Xu, J. Chang, Bioglass Activated Albumin Hydrogels for Wound Healing, Advanced Healthcare Materials 7(16) (2018) 1800144. [7] C. Yang, R. Xue, Q. Zhang, S. Yang, P. Liu, L. Chen, K. Wang, X. Zhang, Y. Wei, Nanoclay cross-linked semi-IPN silk sericin/poly(NIPAm/LMSH) nanocomposite hydrogel: An outstanding antibacterial wound dressing, Materials Science and Engineering: C 81 (2017) 303-313. [8] R.J. 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Degreef, J.J. Vranckx, M. Garmyn, P. Massage, M. van Brussel, Skin grafting and wound healing-the "dermato-plastic team approach", Clinics in dermatology 23(4) (2005) 343-52. [14] E.Z. Browne, Jr., Complications of skin grafts and pedicle flaps, Hand clinics 2(2) (1986) 353-9. [15] R. Shimizu, K. Kishi, Skin graft, Plast Surg Int 2012 (2012) 563493-563493. [16] H.P. Ehrlich, T.K. Hunt, Collagen Organization Critical Role in Wound Contraction, Advances in wound care 1(1) (2012) 3-9. [17] J. Huang, J. Ren, G. Chen, Z. Li, Y. Liu, G. Wang, X. Wu, Tunable sequential drug delivery system based on chitosan/hyaluronic acid hydrogels and PLGA microspheres for management of non-healing infected wounds, Materials science & engineering. C, Materials for biological applications 89 (2018) 213-222. [18] J.S. Boateng, K.H. Matthews, H.N. Stevens, G.M. Eccleston, Wound healing dressings and drug delivery systems: a review, Journal of pharmaceutical sciences 97(8) (2008) 2892-923. [19] N.J. Percival, Classification of Wounds and their Management, Surgery - Oxford International Edition 20(5) (2002) 114-117. [20] M.P. Ribeiro, P.I. Morgado, S.P. Miguel, P. Coutinho, I.J. Correia, Dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing, Materials science & engineering. C, Materials for biological applications 33(5) (2013) 2958-66. [21] G.S. Lazarus, D.M. Cooper, D.R. Knighton, D.J. Margolis, R.E. Pecoraro, G. Rodeheaver, M.C. Robson, Definitions and guidelines for assessment of wounds and evaluation of healing, Archives of dermatology 130(4) (1994) 489-93. [22] S.R. Van Tomme, M.J. van Steenbergen, S.C. De Smedt, C.F. van Nostrum, W.E. Hennink, Self-gelling hydrogels based on oppositely charged dextran microspheres, Biomaterials 26(14) (2005) 2129-2135. [23] E.M. Ahmed, Hydrogel: Preparation, characterization, and applications: A review, Journal of Advanced Research 6(2) (2015) 105-121. [24] H. Huang, X. Qi, Y. Chen, Z. Wu, Thermo-sensitive hydrogels for delivering biotherapeutic molecules: A review, Saudi Pharmaceutical Journal (2019). [25] I. Kurniawansyah, I. Sopyan, A. Aditya Wahyu, H. Nuraini, D. Alminda Fikri, N. Anggun, PREFORMED GEL VS IN SITU GEL: A REVIEW, 2018.

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Journal Pre-proof (TOCN:PVA:Cu/Plu) Weight Ratio (g/ml)

Sample Name

(TOCN:PVA:Cu/Plu) Percent Ration (%)

TOCN-5PVACu

0.01:0.05:0.0074:0.005

1:5:0.74:0.5

TOCN-7.5PVACu

0.01:0.075:0.0074:0.005

1:7.5:0.74:0.5

TOCN-10PVACu

0.01:0.1:0.0074:0.005

1:10:0.74:0.5

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Table

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Journal Pre-proof

Credited Author Statement

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1. Anha Afrin Shefa has conducted hydrogel fabrication, characterization, in vitro study, in vivo study and written the whole manuscript. 2. Tamanna Sultana played a vital role in in vivo study and checked manuscript. 3. Myeong Ki Park conducted western blot study and checked manuscript. 4. Sun Young Lee and Jae-Gyoung Gwon have synthesized TEMPO-oxidized cellulose for hydrogel preparation. 5. Byong-Taek Lee helped in idea generation, raw data management, manuscript writing.

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Journal Pre-proof Graphical abstract

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Highlights:  Curcumin incorporated into oxidized cellulose-polyvinyl alcohol hydrogel system by freeze-thawing process.  Desired viscosity ensures injectibility of hydrogel.  L929 fibroblast cells uptake curcumin-pluronic micelle when released from hydrogel system.  Curcumin incorporation accelerates full-thickness skin wound healing.

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