Basu2018 in Vitro and in Vivo Evaluation of The Wound Healing Properties of Nanofibrillated Cellulose Hydrogels. ACS Applied Bio Materials

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Article
In vitro and in vivo evaluation of the wound healing
properties of nanofibrillated cellulose hydrogels
Alex Basu, Gunta Celma, Maria Strømme, and Natalia Ferraz
ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00370 • Publication Date (Web): 12 Nov 2018
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Page 1 of 27 ACS Applied Bio Materials
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4 In vitro and in vivo evaluation of the wound healing properties of
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6 nanofibrillated cellulose hydrogels
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9 Alex Basu, Gunta Celma, Maria Strømme and Natalia Ferraz*
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Nanotechnology and Functional Materials, Department of Engineering Sciences, Uppsala University,
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14 Box 534, 751 21 Uppsala, Sweden.
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* Corresponding author: natalia.ferraz@angstrom.uu.se
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20 Abstract
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Current trends in wound care research move towards the development of wound healing dressings designed
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25 to treat different types of wounds (e.g. burns and chronic wounds) and towards tailoring treatments for
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27 different stages of the wound healing process. In this context, the development of advanced nanotherapeutic
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29 materials is highlighted as a promising strategy to efficiently control specific phases of the wound healing
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31 process. Here, Ca2+-crosslinked wood-derived nanofibrillated cellulose (NFC) hydrogels are evaluated as
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33 wound healing dressings. In vitro biocompatibility assays were performed to study the interaction of the
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35 NFC hydrogels with cellular processes that are tightly related to wound healing. Moreover, an in vivo dermo-
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37 epidermic full thickness wound healing model in rat was used to uncover the wound healing ability of the
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Ca2+-crosslinked NFC hydrogels. The in vitro experiments showed that the NFC hydrogels were able to
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support fibroblast and keratinocyte proliferation. A potential effect of the hydrogels on triggering
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44 keratinocyte differentiation was furthermore proposed. In vivo, the NFC hydrogels stimulated healing
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46 without causing any adverse local tissue effects, potentially owing to their moisture-donating properties and
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48 the herein discussed aiding effect of the Ca2+-crosslinker on epidermal generation. Thus, this work
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50 extensively demonstrates the wound healing ability of NFC hydrogels and presents an important milestone
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52 in the research on NFC towards advanced wound healing applications.
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55 Keywords: nanocellulose, ion-crosslinking, keratinocytes, fibroblasts, scratch test, in vivo
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60 ACS Paragon Plus Environment
ACS Applied Bio Materials Page 2 of 27
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1 Introduction
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6 Cutaneous wound healing is a highly coordinated process assembled by the consecutive, yet overlapping,
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8 phases of hemostasis, inflammation, proliferation and remodeling.1 Disruption to this progression may lead
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10 to the development of slow- or non-healing wounds that places great stress on patients and health care alike.
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12 One approach to finding treatments for these wounds relates to the development of advanced
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14 nanotherapeutic materials that efficiently control specific parts of the wound healing process.2 Such
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development is made possible by the ability to tune a nanomaterial’s physicochemical properties (e.g. size,
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colloidal stability, surface charge, surface functionalization and hydrophilicity) in order to influence the
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21 outcome of the wound healing process, a possibility not presented by their bulk counterparts.2-3 Examples
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23 of nanotherapeutic materials include materials that function as grafts or bioengineered skin substitutes;4-5
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25 materials that can deliver growth factors for enhanced tissue regeneration;6 nanoparticles with, e.g.,
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27 antibacterial, anti-inflammatory or otherwise healing enhancing properties;7-9 and dressing materials that
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29 create an optimal healing environment for certain types of wounds.10
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32 Wood-derived nanofibrillated cellulose (NFC) is a great example of a nanomaterial with highly tunable
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34 properties. Typically, NFC materials consist of fibrils with a diameter of 3-5 nanometers and a length of up
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36 to several micrometers that can form aggregates of 20-50 nanometer thickness depending on the production
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38 process.11 By modifying the physicochemical properties and synthesis approach of NFC materials, various
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forms (e.g. films, aerogels, gel suspensions and hydrogels) with versatile properties are obtainable. This has
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created an interest in utilizing NFC to produce novel nanotherapeutic materials for advanced wound care
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45 applications.12-15 The sustainability aspect and abundancy of NFC also make it of great societal
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47 importance.11, 16-18
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50 Hydrogels consist of water-swollen, crosslinked, polymeric networks that form insoluble and hydrophilic
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52 self-standing gels.19 They display many desirable properties of a wound healing dressing. The capability of
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54 hydrogels to donate fluid makes them optimal for maintaining a moist wound bed, which aids the autolytic
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56 debridement of necrotic tissue and epidermal migration.10, 20-21 Furthermore, hydrogels are non-adherent to
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3 tissue and provide a cooling sensation for the patient, which has been shown to reduce pain during
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5 treatment.22
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8 The possibility to form hydrogels of NFC functionalized with negatively charged carboxyl groups, by the
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10 simple and reaction free method of ion-crosslinking, was introduced by Dong and co-workers.23 In their
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study, the mechanism of gelling was attributed to mainly two phenomena: the screening of the interfibril
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repulsive forces generated by the carboxylated NFC surface which enables fibril entanglement and weak
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17 interactive forces such as hydrogen bonding and van der Waals interactions between the fibrils; and more
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19 dominantly, by interfibril crosslinking facilitated by the specific interaction between carboxyl groups of
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21 neighboring fibrils and the polycation. The suitability of employing Ca2+-crosslinked NFC hydrogels for the
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23 development of advanced wound care dressings - in terms of beneficial physicochemical properties,
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25 biocompatibility and hemocompatibility - was later shown by us.24-25 The studied Ca2+-crosslinked NFC
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27 hydrogel consists of an entangled fibrous network and exhibits a solid-like behavior with a storage modulus
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29 of 24.9 ± 4.4 kPa, and a good moisture retention capability that affords it the ability to maintain a moist
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wound bed.24-26 Moreover, in vitro investigations showed that the hydrogel are non-toxic to dermal and
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immune cells,24 inert in terms of inflammatory response24-25 and able to act as an effective hemostatic
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36 dressing.25 We further demonstrated the ability to tailor the antibacterial properties of this class of material
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38 by changing the crosslinking ion,27 and explored the use of the Ca2+-crosslinked NFC hydrogel as a protein
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40 carrier.26 Recently, Liu et al. investigated the drug delivery and wound healing capability of
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42 polydopamine/NFC composite hydrogels with successful outcome.28 In another study, NFC/gelatin/silver
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44 composite hydrogels were probed as antibacterial wound dressings.29 However, while NFC hydrogels and
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46 composites thereof have been proposed for various wound healing applications (including wound dressings,
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48 drug delivery vehicles and 3D-printed scaffolds for advanced skin regeneration),16, 30-31 we are not aware of
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50 any comprehensive study on the in vivo wound healing properties of the Ca2+-crosslinked NFC hydrogel.
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53 The present study adds to our previous work and aims at investigating the specific effects of a Ca2+-
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crosslinked NFC hydrogel on dermal and epidermal cell migration and proliferation, which are essential to
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3 the granulation and re-epithelization of wounds. Human fibroblasts and keratinocytes are herein employed
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5 in in vitro migration and proliferation assays. While the charged surface of the NFC hydrogel, as well as the
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7 presence of calcium, may alone affect cell behavior, a composite Ca2+-crosslinked NFC-collagen hydrogel
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is also investigated to explore the possibility of tuning the material’s interactions with these specific cells.
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12 Furthermore, to uncover the in vivo wound healing ability of the Ca2+-crosslinked NFC hydrogel, a dermo-
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14 epidermic full thickness wound model in rat is employed. The results presented herein are foreseen to
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16 significantly advance the research on the use of NFC for advanced wound healing applications.
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20 2 Materials and Methods
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2.1 Chemicals and reagents
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Unmodified biocide-free NFC prepared by enzymatic pretreatment of bleached sulfite softwood pulp
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28 (Dissolving Plus, Domsjö fabriker AB, Sweden) as described by Pääkkö et al.32 was provided by RISE
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30 Bioeconomy (Sweden). 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), Ca(NO3)2, NaBr, NaClO, NaOH,
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32 ethanol, mitomycin C (MMC) and calcein-AM were obtained from Sigma-Aldrich (USA). Alamar Blue
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34 (AB) reagent was purchased from Invitrogen (USA). Dulbecco's Modified Eagle Medium: Nutrient Mixture
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36 F-12 (DMEM-F12), Keratinocyte-Serum Free Medium (KSFM) and Dulbecco’s Phosphate Buffered Saline
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38 (PBS) were obtained from Thermo Fisher Scientific (USA).
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41 2.2 Preparation of materials
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44 2.2.1 Preparation of anionic NFC
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46 TEMPO-mediated oxidation (TEMPO, NaBr, NaClO, pH 10.3) of unmodified NFC was done as previously
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48 described.24 The reaction was performed at room temperature for 2 h and was quenched by the addition of
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1% (v/v) of ethanol. Resulting anionic NFC dispersion was purified by dialysis against deionized water and
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concentrated to 3% (w/w) dry content by gently heating the dispersion under stirring to avoid agglomeration
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55 of the nanofibrils. The carboxyl group content was determined to be 1482 ± 108 µmol/g by performing
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1
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3 conductometric titration following the protocol described by Hua et al,33 and the zeta potential was -35 ± 7
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5 mV. The anionic NFC used to produce NFC hydrogels for the in vivo studies was prepared and purified
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7 under clean conditions with ultrapure water to minimize endotoxin contamination.
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10 2.2.2 Preparation of NFC hydrogels
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NFC and NFC-collagen hydrogels were prepared as previously described.25 A NFC-collagen dispersion was
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made by adding bovine type-1 collagen (2% (w/w) swollen collagen dispersion in water) to 3% (w/w)
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17 anionic NFC at a final concentration of 0.1% (w/w). The mix was homogenized by high-energy
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19 ultrasonication (Vibracell 600 W, USA). Anionic NFC (3% w/w) and NFC-collagen dispersions were
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21 separately added to molds and crosslinked by Ca(NO3)2 (100 mM final concentration) to form hydrogels of
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23 1 mm thickness. After 24 h, unbound Ca2+ was removed by washing the hydrogels with deionized water.
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25 Hydrogels were sterilized by UV irradiation. NFC hydrogels for the in vivo experiments were prepared
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27 separately under clean conditions using ultrapure water to minimize the presence of endotoxins. After UV
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29 sterilization, the hydrogels were packed in air-tight sterile aluminum containers until use. The endotoxin
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level of the TEMPO-oxidized NFC used for the production of the hydrogels was 187 EU/g cellulose, while
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the final endotoxin concentration of the NFC hydrogel was 0.4 EU/g hydrogel (corresponding to 0.5
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36 EU/device), as determined by the Pyrochrome test (Associates of Cape Cod, USA). Thus, the NFC
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38 hydrogels used for the in vivo test complied with FDA regulations on medical devices (<20 EU/device).34
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41 2.3 In vitro cell studies
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43 Human dermal fibroblasts (hDF) (European Collection of Authenticated Cell Cultures) cultured in DMEM-
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45 F12 containing 10% (v/v) fetal bovine serum (FBS), 100 IU/ml penicillin, 100 µg/ml streptomycin and 20
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47 mM L-glutamine, and human keratinocyte cell line CCD 1106 KERTr (ATCC® CRL2309™) (KERT)
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49 (American Type Culture Collection) cultured in KSFM supplemented with 0.05 mg/ml bovine pituitary
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51 extract (BPE), 35 ng/ml epidermal growth factor (EGF), 100 IU/ml penicillin and 100 µg/ml streptomycin,
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were used as model cells. The cell cultures were kept in a 37 °C and 5% CO2 incubator with humidified
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56 atmosphere. The commercial hydrofiber wound dressing Aquacel Extra (ConvaTec, UK) comprised of
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3 carboxymethyl cellulose was used as reference material since in a water-swollen state it resembles the
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5 studied NFC-based hydrogels in terms of hydration and material composition.
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8 2.3.1 Cell migration
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10 An in vitro scratch assay described by Liang et al.35 was used to evaluate fibroblast and keratinocyte
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migration in the presence of the NFC-based hydrogels. hDF cells were seeded into a 24-well plate at a
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concentration of 50,000 cells/well in complete DMEM-F12 culture medium and incubated at 37 °C and 5%
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17 CO2 for 24 h to form confluent monolayers. The hDF monolayers were scratched with a 1 ml pipette tip and
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19 washed with PBS. DMEM-F12 supplemented with 1% (v/v) FBS, 100 IU/ml penicillin, 100 µg/ml
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21 streptomycin and 20 mM L-glutamine was added to the wells and 0 h reference images of the scratch widths
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23 were obtained by light microscopy (Nikon ECLIPSE TE2000-U, Japan). The NFC hydrogel, the NFC-
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25 collagen hydrogel and rehydrated Aquacel Extra were added to cover the scratched hDF monolayers.
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27 Scratched monolayers in wells without any dressing material were the control. The migration of hDF cells
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was imaged at 12 h and 24 h by light microscopy (Nikon ECLIPSE TE2000-U, Japan). Each sample was
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run in triplicate and each experiment was repeated three times.
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KERT cells were seeded into a 24-well plate at a concentration of 60,000 cells/well in complete KSFM
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37 culture medium and incubated at 37 °C and 5% CO2 for 24 h to form confluent monolayers. The culture
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39 medium was exchanged for KSFM-MMC medium (containing 0.05 mg/ml BPE, 35 ng/ml EGF, 100 IU/ml
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41 penicillin, 100 µg/ml streptomycin and 1 µg/ml MMC) and the cells were incubated for an additional 24 h
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43 at 37 °C and 5% CO2 for proliferation to halt. Cell monolayers were scratched, washed with PBS and fresh
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45 KSFM-MMC medium was added. KERT migration in the presence of the NFC hydrogel, the NFC-collagen
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47 hydrogel and Aquacel Extra was thereafter evaluated in the same way as in the hDF experiments.
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50 From the light microscopy images, migration at 12 h and 24 h in terms of percentage of the initial scratch
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52 width was calculated. Monolayer scratches at 0 h, 12 h and 24 h were stained by incubating the cells with
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300 µl of culture medium containing 0.1% (w/w) of calcein-AM for 15 min at 37 °C and 5% CO2.
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3 Thereafter, viable cells were imaged using a fluorescence microscope (490 nm excitation, 515 nm emission)
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5 (Nikon ECLIPSE TE2000-U, Japan).
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8 2.3.2 Cell-hydrogel interactions
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The NFC hydrogel, the NFC-collagen hydrogel and rehydrated Aquacel Extra were placed to cover the
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13 bottom of the wells of a 96-well plate. The samples were allowed to soak in complete culture medium
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15 (DMEM-F12 and KSFM for hDF and KERT cells, respectively) while cell suspensions were prepared. The
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17 soaking medium was removed and cells (5000 cells/well (hDF) or 6000 cells/well (KERT)) suspended in
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19 200 µl of respective complete culture medium were seeded onto the samples. Cells cultured on tissue culture
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21 plate (TCP) served as control. Viability and morphology of hDF cells were assessed at day 1, 3 and 7 while
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23 KERT viability and morphology were assessed at day 1, 2 and 3. Each sample was run in triplicate and each
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25 experiment was repeated five times.
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28 Metabolic activity was determined by the AB assay. At the respective time points, culture supernatants were
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30 replaced with 200 µl of AB stock solution diluted 1:10 in complete cell culture medium (DMEM-F12 and
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32 KSFM for hDF and KERT cells, respectively) and the cells were incubated with the AB reaction mix for
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120 min at 37 °C and 5% CO2. Aliquots of 100 µl from each well were transferred to a black 96-well plate
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37 and the fluorescence intensity was measured by a microplate reader (Tecan Infinite M200, Switzerland) at
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39 560 nm excitation and 590 nm emission wavelength. Cell morphology of viable adherent cells at the
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41 respective time points was assessed by staining adherent cells by the addition of 200 µl of culture medium
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43 containing 0.1% (w/w) calcein-AM and incubation at 37 °C and 5% CO2 for 15 min. The stained cells were
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45 imaged in the same way as in the migration assay.
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48 The effect of each component of the NFC hydrogel on KERT cells was investigated by culturing the cells
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50 in complete KSFM medium supplemented with 1.2 mM of Ca2+ (crosslinking ion) and in complete medium
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52 supplemented with 500 µg/ml of non-crosslinked anionic NFC suspension. After 72 hours, cell culture
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3 medium was removed and the cells were stained with calcein-AM and imaged as previously described. Cells
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8 2.4 In vivo wound healing model
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11 2.4.1 Experimental design and surgical procedure
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13 The in vivo wound healing study was conducted according to the ISO 10993-6:2007 standard36 at NAMSA
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15 contract research laboratory (France) and with animal management conditions conforming to the European
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requirements (Directive EU/2010/63).
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A total of 6 Sprague Dawley rats (Charles River Laboratories, France), of weight 324-348 g at wound
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creation, were used. The rats were kept separately in shoebox cages at room temperature (20 – 24 °C) and
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25 under controlled light cycles (12 h light, 12 h dark). On the day of wound creation (day 0), each rat was
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27 injected subcutaneously with an analgesic (buprenorphine, Buprecare®, Axience, France) and anesthetized
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29 by inhalation of isoflurane (IsoFlo®, Axience, France). The fur was clipped from the surgical area. A scalpel
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31 was used to surgically induce two paravertebral dermo-epidermic wounds (2.5 cm x 2.5 cm), preserving the
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33 panniculus carnosus, one on each side of the back of each rat. The NFC hydrogel and the control dressing
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35 comprised of saline soaked sterile non-woven cotton gauze (Sylamed, France), were cut into pieces of 3 cm
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37 x 4 cm. The absence of bleeding of the wound was checked carefully before applying the NFC hydrogel
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and the control dressing to one wound each on each rat (n=6 sites per dressing). The primary dressings were
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covered by a secondary dressing composed of sterile semi-permeable polyurethane adhesive dressing
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44 (Tegaderm®, 3M, USA), sterile woven gauze (Sylamed, France) and elastic adhesive bandage
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46 (Urgostrapping®, Urgo, UK). The rats were moved to a recovery area and monitored for recovery from the
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48 anesthetic until sternal recumbency was achieved. After recovery, each rat was returned to its cage and
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50 observed for general health. Three times a week, the dressings were carefully changed and macroscopic
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52 evaluations of wounds and dressings were done (see sections below). Prior to re-application of dressings,
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54 the rats were weighed, wounds were washed with lukewarm saline solution and macroscopic pictures of the
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56 wounds were taken. At each dressing change, volatile anaesthesia (isoflurane, IsoFlo®, Axience, France)
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3 was used. At day 25, rats were euthanized by an intravenous injection of a lethal solution (DolethalND,
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5 Vetoquinol, Sweden) and the wounds were sampled for histopathological analysis.
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8 2.4.2 Macroscopic evaluation of the dressings
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10 At every dressing change the adherence of dressings to wounds and the structural stability of the dressings
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were evaluated according to a grading score (Table S1).
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15 2.4.3 Macroscopic evaluation of the wounds
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Wound evaluation included observation and scoring (Table S1) of peri-wound skin status, presence of
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20 exudate, presence of blood, presence of fibrin, presence of granulation tissue, signs of maceration and/or
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22 infection and re-epithelialization at every dressing change. Additionally, the evolution of the non-
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24 epidermized wound surfaces (percentage of surface uncovered with epithelium) was evaluated by
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26 quantitative morphometric analysis performed on the macrophotographs of each wound taken at every
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28 dressing change. The data analysis was conducted using a morphometry software (Perfect Image® 7.5, Clara
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30 Vision). At each time point, the surface of each wound was expressed in cm² and as a percentage of the
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32 initial wound surface (residual wound surface). The 25, 50, 75 and 95% healing times were calculated
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34 (graphical interpolation) and defined as the mean time required to achieve 25, 50, 75 or 95% of the healing
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of the initial wound surface.
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2.4.4 Histopathological evaluation
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At the end of the study, for each rat, the wounds were sampled at full depth including a margin of at least 5
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44 mm of healthy uninjured skin around each original wound. Each sample was fixed in 10% neutral buffered
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46 formalin solution and subsequently dehydrated in alcohol solutions of increasing concentration, cleared in
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48 xylene and embedded in paraffin. Two central sections were cut with a microtome (4.5 µm thickness). One
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50 section was stained with a modified Masson’s trichrome (MT) and another with safranin-hematoxylin-eosin
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52 (SHE). Qualitative and semi-quantitative histopathological evaluation of the local tissue effects at the wound
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54 location and overall wound healing performance were conducted according to the ISO 10993-6 standard.36
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3 The test score systems employed for the qualitative and semi-quantitative histopathological evaluation are
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5 presented in Table S2 and Table S3.
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8 2.5 Statistical analysis
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10 In vitro data was analyzed by one-way analysis of variance and Fisher’s least significant difference with
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Bonferroni’s post-hoc test using R Studio v. 0.99.489. Values at p < 0.05 were considered statistically
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different. Statistical analysis of data from the in vivo study was conducted using non-parametric tests with
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17 a 5% risk using SPSS® v. 24.0.
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21 3 Results and Discussion
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24 3.1 In vitro cell migration
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26 Major cell types of the dermis and epidermis include fibroblasts and keratinocytes, respectively. Besides
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providing vital barrier properties that protect the host, these cells are essential to the repair of skin
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31 subsequent to injury.37-40 Proliferation and migration of fibroblasts and keratinocytes play a key role during
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33 the granulation and re-epithelialization of wounds,41 thus making any interactions between dressing
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35 materials and such processes an important aspect to consider in the material evaluation toward wound
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37 healing applications. In this study, human dermal and epidermal cells were used to investigate the in vitro
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39 effect of Ca2+-crosslinked wood-derived NFC-based hydrogels on skin cell migration, adhesion,
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41 proliferation and morphology as a step forward in the biocompatibility evaluation of the materials toward
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43 advanced wound healing applications. Two types of NFC-based hydrogels were used: a hydrogel containing
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45 anionic NFC and a hydrogel consisting of a mixture of anionic NFC and type-1 collagen. Inclusion of type-1
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collagen was of interest due to its acknowledged control of cellular functions of fibroblasts and keratinocytes
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50 (e.g. cell shape, adhesion, differentiation and migration)42 and overall positive effect on wound healing by
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52 enhancing re-epithelialization and reducing scar formation.43 In our previous work, scanning electron
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54 microscopy investigations demonstrated that the collagen was evenly distributed within the NFC matrix,
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56 and that this composite hydrogel exhibited enhanced mechanical stability compared to the pure NFC
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1
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3 hydrogel as evidenced by the increase in storage modulus from 26.4 to 36.2 kPa (obtained by rheological
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5 measurements at a strain of 0.5% and frequency of 80 Hz).
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8 The use of the scratch assay as an in vitro wound healing model is a great first-choice method to investigate
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10 the potential of novel wound healing dressings due to the ethical concerns of employing in vivo models for
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early screenings of such materials. Results of hDF migration under both NFC-based hydrogels and the
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15 commercial dressing Aquacel Extra showed that all studied materials had a similar effect on cell migration
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17 at 12 h, while over 24 h the NFC-collagen hydrogel promoted faster hDF migration compared to the NFC
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19 hydrogel (Figure 1a). KERT migration under the NFC-collagen hydrogel was marginally faster than for the
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21 NFC hydrogel after 24 h, and significantly faster than for the Aquacel Extra group during both measured
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23 time points (Figure 1b). Thus, the aiding effect of the NFC-collagen hydrogel appeared stronger on
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25 keratinocytes than fibroblasts. This observation is supported by previous work where type-1 collagen is
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described as a prominent promotor of keratinocyte migration, a trait linked to the 11 integrin receptor of
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30 the keratinocyte cell.44-45 For both model cells, migration in the absence of dressing materials was generally
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32 faster than migration under said materials. This could be a limitation of the in vitro model due to physical
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34 hindrance of cell migration in the presence of a material on top of the cell monolayer. An additional
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36 limitation when investigating hydrogels using this model is that the proposed beneficial effect of the highly
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38 hydrated matrix of hydrogels on facilitating cell migration46-47 is limited by the presence of cell culture
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40 medium.
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21 Figure 1. Migration of a) hDF and b) KERT cells at 12 h and 24 h subsequent to the scratching of cell monolayers and
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23 application of NFC hydrogel, NFC-collagen hydrogel and the reference material Aquacel Extra to cover the scratch.
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25 Cells migrating on tissue culture plate (TCP) in the absence of a dressing material was the control. Data represents
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27 mean ± standard error of the mean for n = 3. Asterisk brackets indicate statistically significant differences (p < 0.05)
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29 between 24 h data points.
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32 Fluorescence microscopy images of viable hDF (Figure 2a) and KERT (Figure 2b) cells at 0 h, 12 h and 24
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34 h after scratching of monolayers display the cell migration and show that the number of viable cells after
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36 removal of the hydrogels was similar to the cell pattern observed in the control, thus giving an indication
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38 that the hydrogels did not affect cell viability adjacent to the scratched area throughout the experiment.
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40 Furthermore, from these images it can be seen that the NFC-based hydrogels did not damage in vitro dermal
41
42 and epidermal cell monolayers after removal. Thus, non-adherent properties of the hydrogels to cell
43
44 monolayers were demonstrated, which is an important characteristic of a wound dressing, as tissue adherent
45
46 dressings can cause damage and pain upon removal.10
47
48
49
50
51
52
53
54
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58 12
59
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31
32
33
34
35
36
Figure 2. Fluorescence microscopy images of calcein-AM stained a) hDF and b) KERT cells at 0 h, 12 h and 24 h
37
38
subsequent to the scratching of cell monolayers and application of the NFC hydrogel, the NFC-collagen hydrogel and
39
40
the reference material Aquacel Extra to cover the scratch. Cells migrating on tissue culture plate (TCP) in the absence
41
42
43 of a dressing material was the control. Scale bar represents 400 µm.
44
45
3.2 In vitro cell-hydrogel interactions
46
47
48
To investigate the effect of the NFC-based hydrogels on hDF and KERT adhesion, proliferation and
49
50 morphology, cells were seeded on the materials and cell characteristics were studied by metabolic activity
51
52 measurements and fluorescence microscopy after 1, 3 and 7 days for hDF and 1, 2 and 3 days for KERT
53
54 cells. hDF cells cultured on the NFC hydrogel, the NFC-collagen hydrogel and Aquacel Extra displayed a
55
56 significantly lower initial adhesion compared to cells on TCP, as indicated by the metabolic activities at day
57
58 13
59
1
2
3 1 (Figure 3a). However, cell proliferation took place as shown by the significant increase in metabolic
4
5 activities with time. At day 7, the percentage of cell number with respect to day 1 represented 380 ± 32, 426
6
7
± 25 and 394 ± 43% for NFC, NFC-collagen and Aquacel Extra, respectively, while the value found for
8
9
10 TCP was 248 ± 48%.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Figure 3. Metabolic activity of a) hDF cells after 1, 3 and 7 days and b) KERT cells after 1, 2 and 3 days subsequent
30
31
to seeding onto the NFC hydrogel, the NFC-collagen hydrogel, Aquacel Extra and tissue culture plate (TCP). Data
32
33
34 represents mean ± standard error of the mean for n = 5. Asterisk indicates a statistically significant difference (p <
35
36 0.05) from the previous time point.
37
38
The fluorescence microscopy images depicted in Figure 4a confirm the low initial adhesion of the hDF cells
39
40
to the NFC-based hydrogels, showing also a spheroid morphology of the cells. However, a gradual recovery
41
42
43 of the hDF cells (Figure 4a), as indicated by the morphological evolution from the spheroid shape at day 1
44
45 to a healthy elongated shape with visible extensions from day 3 and forward was observed. Thus, after an
46
47 adaptation period on the NFC surface, cells were able to spread and proliferate reaching confluency after 7
48
49 days of culture. The increasing hDF cell densities on the NFC hydrogel, the NFC-collagen hydrogel and
50
51 TCP seen in the fluorescence microscopy images furthermore correlated with the metabolic activity data
52
53 (Figure 3a). Note that representative microscopy images of cells cultured on Aquacel Extra could not be
54
55
obtained due to the opaque nature of this material.
56
57
58 14
59
1
2
3 In general, there are several factors that can affect cell adhesion to a material surface. Cell-surface
4
5 interactions are ultimately determined by interactions between cells and surface bound proteins.48 Thus, a
6
7 correlation between protein adsorption and cell adhesion is often observable. Physical adsorption of proteins
8
9
on hydrogel surfaces is expected to be prevented by the low interfacial energy between the highly hydrated
10
11
12 gel phase and the aqueous biological medium,49 and as a consequence poor cell adhesion on hydrogel
13
14 surfaces is anticipated. Nevertheless, there are a number of parameters that can change this behavior of
15
16 hydrogels, with material characteristics such as surface wettability, presence of functional groups, surface
17
18 group density, stiffness and roughness, being recognized as pivotal.47-48, 50-52 As an example, Faucheux et al.
19
20 previously demonstrated that the presence of carboxyl groups on a material surface may improve cell
21
22 adhesion.53 In accordance, Rashad et al. presented carboxylated NFC hydrogels as better candidates for
23
24 tissue engineering applications than the carboxymethylated variant in terms of cell adhesion, and highlighted
25
26 the significance of surface chemistry for the interactions between fibroblast cells and the NFC hydrogel.46
27
28
Based on these reports, the herein studied NFC-based hydrogels containing carboxyl groups could be
29
30
31 expected to exhibit a favorable chemistry for fibroblast adhesion. From the obtained results however, it is
32
33 shown that an individual parameter like hydrogel surface chemistry does not alone affect cell adherence
34
35 properties, and physicochemical differences (e.g. hydrogel stiffness) as a consequence of changes in
36
37 composition are likely to contribute to variations in fibroblast adherence between the studies.
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 15
59
1
2
3 Figure 4. Fluorescence microscopy images of calcein-AM stained (a) hDF cells after 1, 3 and 7 days, and (b) KERT
4
5 cells after 1, 2 and 3 days subsequent to seeding onto the NFC hydrogel, the NFC-collagen hydrogel and tissue culture
6
7 plate (TCP). Scale bar represents 200 µm. Note the confluency and normal elongated morphology of hDF cells on both
8
9 hydrogels at day 7 and the fused KERT morphology on both hydrogels at day 2 and 3.
10
11
12 The number of initial adherent KERT cells on the NFC-based hydrogels was comparable to the number of
13
14 cells on the TCP control and they proliferated in a similar way as shown by the metabolic activity data
15
16 (Figure 3b). At day 3, the percentage of cell number with respect to day 1 represented 339 ± 21, 291 ± 22
17
18 and 375 ± 16% for NFC, NFC-collagen and TCP, respectively. KERT cells on Aquacel Extra displayed
19
20 poor adhesion and no significant proliferation was observed as indicated by the non-significant change in
21
22 cell metabolic activity over time (Figure 3b). Fluorescence microscopy images of adherent KERT cells
23
24
showed that the NFC hydrogel and the NFC-collagen hydrogel promoted a marked morphological change
25
26
27 of the KERT cells compared to cells grown on TCP (Figure 4b). Islands of fused cells could already be
28
29 observed at day 1, and at day 2 and 3 they had significantly increased in size. On TCP, KERT cells displayed
30
31 a typical polygonal morphology at all time points. The increase in the size of KERT islands on the NFC
32
33 hydrogel and the NFC-collagen hydrogel as well as the increased number of KERT cells on TCP correlate
34
35 with measured metabolic activities (Figure 3b).
36
37
38 A spread and fused morphology of keratinocytes has been described as a sign of keratinocyte differentiation,
39
40 a process that is tightly regulated by, amongst other mechanisms, extracellular calcium concentration.54-57
41
42 Thus, we investigated if the Ca2+ crosslinking ion of the NFC-based hydrogels could be responsible for the
43
44 observed changes in KERT morphology. As shown in Figure 5, results indicated that KERT cells in the
45
46 presence of the crosslinking ion underwent cell fusion, while KERT cells cultured with the non-crosslinked
47
48
NFC suspension and on TCP maintained an individualized and polygonal morphology.
49
50
51
52
53
54
55
56
57
58 16
59
1
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3
4
5
6
7
8
9
10
11
12
13
14 Figure 5. Fluorescence microscopy images of KERT cells after 3 days incubation with Ca2+ (1.2 mM) and non-
15
16 crosslinked NFC suspension (500 µg/ml). The control was KERT cells cultured on tissue culture plate (TCP) for the
17
18 same time period. Scale bar represents 100 µm. Note the KERT cell fusion (as indicated by white arrows) in the
19
20 presence of Ca2+, while cells cultured in the presence of NFC suspension maintain the same KERT morphology as in
21
22 the control (TCP).
23
24
25 The calcium switch, i.e. an increase in calcium concentration, initiates the differentiation processes in
26
27 keratinocytes and triggers morphological changes and the formation of intracellular contacts important for
28
29 the differentiation process.57 The process continues with the loss of cell growth potential and the sequential
30
31 expression of differentiation markers.58 Unfortunately, there is no data available on the expression of these
32
33 differentiation markers (e.g. involucrin and cytokeratin 10) by the KERT cell line. Our attempts at
34
35 evaluating involucrin and cytokeratin 10 by western blotting of cell lysates did not show upregulation of
36
37 such markers in neither samples nor the positive control (cells were stimulated with conditions similar to
38
39
those that have shown positive results with the widely studied cell line HaCat and primary keratinocytes,54-56
40
41
results not shown). Nevertheless, the herein observed changes in morphology and the previously
42
43
44 demonstrated influence of calcium in keratinocyte differentiation, allow us to hypothesize about a potential
45
46 ability of the NFC-based hydrogels to trigger such differentiation. Calcium-dependent keratinocyte
47
48 differentiation is responsible for the formation of the spinous, granular and cornified layer of the epidermis.59
49
50 As this differentiation is central to re-epithelization, the NFC-based hydrogels may therefore be able to
51
52 augment wound healing by aiding epidermal regeneration.
53
54
55
56
57
58 17
59
1
2
3 The similarity of cell adhesion, morphology and proliferation between the NFC-based hydrogels indicates
4
5 that the presence of collagen within the hydrogel matrix did not significantly affect keratinocyte and
6
7 fibroblast responses despite the hypothesis of such influence.
8
9
10 3.3 In vivo wound healing ability
11
12
The in vitro biocompatibility assays suggested the NFC-based hydrogels as suitable candidates for
13
14
15
continued testing due to their ability to support dermal and epidermal cell proliferation, their non-adhering
16
17 nature to cell monolayers and their potential ability to trigger keratinocyte differentiation. Due to the minor
18
19 differences in performance between the NFC and NFC-collagen hydrogels, the sustainability factor of the
20
21 NFC hydrogel (produced of only anionic NFC) and the risk of immune reactions caused by bovine collagen,
22
23 the NFC hydrogel was deemed a better candidate and thus selected for further evaluation in an in vivo wound
24
25 healing assay. In this experiment, the performance of ultrapure NFC hydrogel dressings was benchmarked
26
27 toward a well-documented treatment comprised of saline soaked gauze commonly used for the management
28
29 of acute wounds.
30
31
32 Dermo-epidermic wounds of size 2.5 cm  2.5 cm allowed comparison of the two treatment methods on the
33
34
same rat. The panniculus carnosus was preserved to limit the contraction associated with wound healing in
35
36
37 rat.60 Throughout the study, the health status of the rats was monitored and individual body weight data was
38
39 recorded. Overall the rats lost weight over the course of the study (-4  2%) (Table S4). This body weight
40
41 loss was attributed to the recovery after wound creation and the repeated anaesthesia and was not linked to
42
43 the treatment methods.
44
45
46 The NFC hydrogel dressing conformed well to the wound bed and adherence to the wound was judged very
47
48 low (Figure S1), an observation that correlates with the in vitro assessment made herein. Moreover, the
49
50 structural stability of the hydrogels remained unchanged during the three-day period they were applied on
51
52 the wounds (Table S5).
53
54
55
56
57
58 18
59
1
2
3 The progression of the healing was registered by macroscopic pictures during 25 days. Representative
4
5 pictures of the wounds at different time points are presented in Figure 6. Macroscopic observation of the
6
7 wounds revealed that the first signs of re-epithelialization (slight to moderate) appeared at day 7 for both
8
9
groups (Figure S2). For the NFC hydrogel dressing, a regular increase of re-epithelialization was observed
10
11
12 from day 7 to day 18 and all wounds were fully healed at day 21 (Figure S2). For the control group, the re-
13
14 epithelialization was retarded compared to the NFC hydrogel group and 25 days after wound creation, no
15
16 control wounds were completely healed. There were no signs of adverse local tissue effects (edema and
17
18 erythema), infection or maceration on neither the wounds nor the skin surrounding the wounds in any of the
19
20 groups throughout the study. Besides, no significant blood or exudate presence was noted, which points
21
22 toward a balanced wound environment that lowers the risk of maceration.10 For both treatments, fibrin
23
24 appeared on the wounds from day 4 and increased slightly until day 7, to then decrease to reach zero on day
25
26 11 (Figure S3). As fibrin acts as a provisional matrix for cell migration during wound healing, it is expected
27
28
to be present at early stages of healing.61 Histopathological evaluation however showed a slight grade of
29
30
31 fibrin for control wounds at day 25 (Table S8) which could be a sign of immature healing. The formation
32
33 of granulation tissue occurred early and in a normal fashion for both dressings (Figure S4).39
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52 Figure 6. Representative pictures of wound healing under the control and the NFC hydrogel dressing during 25 days.
53
54 Note the marked accelerated healing of wounds treated with the NFC hydrogel dressing.
55
56
57
58 19
59
1
2
3 Morphometric analysis was conducted to determine the residual wound surface area over 25 days (Figure
4
5 7). The decrease of the residual wound surface area had the same profile between the NFC hydrogel and the
6
7 control treatment but was faster for the NFC hydrogel compared to the control as it had lower residual
8
9
wound surface area at each time point. Statistical differences were observed between the two groups at each
10
11
12 time point from day 7. From day 21, the wound surface area was zero for the NFC hydrogel and
13
14 corresponded to a complete healing of all wounds (full re-epithelialization).
15
16
17 120
Control
18
* NFC hydrogel
19
100
20 *
Residual wound surface (%)
21
22 80
23 *
24
25 60 *
*
26
27 *
40 *
28
29 *
*
30 20
31
32
33 0
34 0 5 10 15 20 25
35 Days
36
37
38 Figure 7. Wound closure as determined by macroscopic evaluation of the residual wound surface area. Data represents
39
mean ± standard error of the mean for n = 6. Asterisk indicates statistically a significant difference (p < 0.05) between
40
41
residual wound surface of wounds under the control and the NFC hydrogel dressing.
42
43
44 The incidence of fully healed wounds for the NFC hydrogel treatment at each time point is presented in
45
46
Table S6. The mean time to achieve 25, 50, 75 and 95% healing for the NFC hydrogel group was 4.7  0.6,
47
48
49 6.7  0.5, 9.8  0.4 and 16.6  0.6 days, respectively (Table S7). Meanwhile, for the control group, the mean
50
51 times were 6.6  1.3, 11.9  1.3 and 19.7  0.8 days to achieve 25, 50 and 75% healing, respectively, and
52
53 no control wounds reached 95% of healing after 25 days.
54
55
56
57
58 20
59
1
2
3 The quality of the healing was determined by histopathological analysis. 25 days after wound creation, the
4
5 NFC hydrogel group presented fully healed wounds with the newly-formed epithelium fully covering the
6
7 granulation tissue of the wound (Figure 8a and c). Results of the semi-quantitative histopathological analysis
8
9
is shown in Table S8. The neo-epithelium was almost similar to the native epithelium present in the adjacent
10
11
12 intact skin and displayed marked to complete signs of differentiation, indentation and dermal adherence. As
13
14 previously discussed, the presence of calcium in the NFC hydrogel could contribute to such proficient re-
15
16 epithelialization. The granulation tissue filling the wound showed the following signs of maturity: absence
17
18 of hemorrhage and fibrin; low numbers of neo-vessels; and low numbers of acute inflammatory cells.39, 62 It
19
20 should be noted that abundant macrophages were still present and appeared filled with residual material of
21
22 either primary or secondary dressing (Figure 8c, marked by blue arrows).
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 Figure 8. Representative photomicrographs of the histopathological evaluation of healing sampled at day 25.
50
51 Photomicrographs a) and c) depict healing of wounds under the NFC hydrogel dressing and b) and d) represent healing
52
53 of wounds under the control dressing. Samples a) and b) were stained with MT and samples c) and d) with SHE. BV:
54
55
56
57
58 21
59
1
2
3 blood vessels, D: dermis, FE: full epithelialization, GT: granulation tissue, PE: partial epithelialization, SC: subcutis,
4
5 blue arrows: filled macrophages. Note the fully healed epithelium on wounds treated with the NFC hydrogel dressing.
6
7
8 For the control group, 25 days after wound creation, all six wounds were incompletely healed with the
9
10 newly-formed epithelium only partially covering the granulation tissue of the wound (Figure 8b and d, Table
11
12 S8). The neo-epithelium on the wound margins displayed moderate differentiation, indentation, and
13
14 adherence to the sub-laying dermis. The granulation tissue filling the wound showed signs of immaturity as
15
16 a presence of hemorrhage and fibrin, together with abundant neo-vessels and acute inflammatory cells were
17
18 noted (Table S8).39, 62 Overall, the histopathological evaluation showed that the NFC hydrogel was not
19
20
associated with any adverse local tissue effect and presented higher healing performance than the control,
21
22
23 as indicated by the fully healed epithelium and the mature granulation tissue.
24
25
26
The collective results herein indicate that the Ca2+-crosslinked NFC hydrogel is able to promote a
27
28 significantly improved wound healing compared to a standardized wet-gauze treatment in an acute wound
29
30 healing model, potentially owing to its previously demonstrated beneficial moisture-donating properties24
31
32 and the herein discussed aiding effect of the Ca2+-crosslinker on epidermal generation. This study therefore
33
34 proves previous hypotheses regarding the wound-healing ability of NFC-based hydrogels. Combined with
35
36 the previously demonstrated ability to act as a barrier against invading pathogens, the tunable bacteriostatic
37
38 properties and the proven hemostatic effect,25, 27 the NFC hydrogel is judged as an appropriate platform-
39
40 material for the continued development of advanced wound healing solutions that efficiently control specific
41
42
parts of the wound healing process.
43
44
45
46
47 4 Conclusions
48
49 In vitro studies showed that Ca2+-crosslinked wood-derived NFC hydrogels supported fibroblast and
50
51 keratinocyte proliferation, and a potential effect of the hydrogels on triggering keratinocyte differentiation
52
53
augmenting to wound healing is proposed. Furthermore, a significant wound healing capability of NFC
54
55
56
hydrogels was demonstrated in an in vivo dermo-epidermic full thickness wound healing model in rat, where
57
58 22
59
1
2
3 the NFC hydrogel promoted faster wound healing than the control treatment. Especially interesting was the
4
5 advanced stage of epidermal re-generation and maturity of the granulation tissue, the non-adherent property
6
7 of the NFC hydrogel, and lack of any adverse local tissue effects as consequence of the NFC hydrogel
8
9
treatment. Thus, this work for the first time thoroughly demonstrates the wound healing ability of NFC-
10
11
12 based hydrogels, and is foreseen to significantly contribute to the research on the use of NFC for wound
13
14 healing applications.
15
16
17
18 5 Supporting Information
19
20
Grading score for macroscopic observations of the dressings and the wounds at each dressing change,
21
22
23 Scoring system used for the semi-quantitative histological evaluation of local tissue effects, Scoring system
24
25 used for the semi-quantitative histological evaluation of the wound healing performance, Scoring system
26
27 used for the semi-quantitative histological evaluation of the wound healing performance, Macroscopic
28
29 evaluation of dressing adherence to wounds, Macroscopic evaluation of the structural stability of dressings,
30
31 Macroscopic evaluation of the re-epithelialization of wounds, Macroscopic evaluation of presence of fibrin
32
33 on wounds, Macroscopic evaluation of granulation tissue formation, Incidence and percentage of fully
34
35 healed wounds per time point, Time to achieve 25, 50, 75 and 95% healing.
36
37
38
39
40 6 Acknowledgements
41
42 We acknowledge the Swedish research council FORMAS for their financial support of this work (Grant
43
44 number 942-2015-475). Dr. Eva Ålander at Rise Bioeconomy, Sweden, is acknowledged for supplying the
45
46 nanofibrillated cellulose. Dr. Levon Manukyan at the Division of Nanotechnology and Functional Materials,
47
48 Uppsala University is acknowledged for fruitful discussions regarding keratinocytes differentiation and
49
50
protein expression.
51
52
53
54
55 7 References
56
57
58 23
59
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Basu2018 in Vitro and in Vivo Evaluation of The Wound Healing Properties of Nanofibrillated Cellulose Hydrogels. ACS Applied Bio Materials

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