Inflammatory responses and tissue reactions to wood-Based nanocellulose scaffolds

Accepted Manuscript Responses and host tissue reactions to wood-based nanocellulose scaffolds

Ahmad Rashad, Salwa Suliman, Manal Mustafa, Torbjørn Ø. Pedersen, Elisabetta Campodoni, Monica Sandri, Kristin Syverud, Kamal Mustafa PII: DOI: Reference:

S0928-4931(18)32413-5 https://doi.org/10.1016/j.msec.2018.11.068 MSC 9088

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

10 August 2018 22 November 2018 27 November 2018

Please cite this article as: Ahmad Rashad, Salwa Suliman, Manal Mustafa, Torbjørn Ø. Pedersen, Elisabetta Campodoni, Monica Sandri, Kristin Syverud, Kamal Mustafa , Responses and host tissue reactions to wood-based nanocellulose scaffolds. Msc (2018), https://doi.org/10.1016/j.msec.2018.11.068

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ACCEPTED MANUSCRIPT

Responses and Host Tissue Reactions to Wood-Based Nanocellulose Scaffolds Ahmad Rashada*, Salwa Sulimana, Manal Mustafab, Torbjørn Ø. Pedersena, Elisabetta Campodonic, Monica Sandric, Kristin Syverudd, e, Kamal Mustafa1a* Department of Clinical Dentistry, University of Bergen, Bergen, Norway

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Oral Health Centre of Expertise in Western Norway, Bergen, Norway

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Institute of Science and Technology for Ceramics, National Research Council of Italy, Faenza, Italy RISE PFI, Trondheim, Norway

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authors:

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*Corresponding

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Department of Chemical Engineering, Norwegian University of Science and Technology, Trondheim, Norway

Ahmad Rashad

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E-mail: [email protected]

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Kamal Mustafa E-mail: [email protected]

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KEYWORDS:

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Phone: +4755586097

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Department of Clinical Dentistry, University of Bergen, Norway

Cellulose Nanofibrils, Inflammation, Cytokines, Macrophages, Degradation, Foreign body reaction

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ACCEPTED MANUSCRIPT Abstract Two wood-derived cellulose nanofibril (CNF) porous scaffolds were prepared by TEMPOoxidation and carboxymethylation. The effects of these scaffolds on the production of inflammatory cytokines by human macrophage-like cells (U937) was profiled in vitro after 1 and

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3 days and in subcutaneous tissues of rats after 4 and 30 days, using PCR and Multiplex arrays.

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Tissue culture plates (TCP) and gelatin scaffolds served as controls in vitro and in vivo respectively.

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After 3 days in vitro, there was no significant difference between the effects of CNF scaffolds and

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TCP on the production of chemokines/growth factors and pro-inflammatory cytokines. At day 4 in vivo there was significantly higher gene expression of the anti-inflammatory IL-1Ra in the CNF

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scaffolds than the gelatin scaffold. Production of IL-1β, IL-6, MCP-1, MIP-1α CXCL-1 and M-

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CSF was significantly less than in the gelatin, demonstrating an early mild inflammatory response. At day 30, both CNF scaffolds significantly stimulated the production of the anti-inflammatory

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cytokine IL-10. Unlike gelatin, neither CNF scaffold had degraded 180 days post-implantation.

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The slow degradation of CNF scaffolds resulted in a foreign body reaction, with high production of IL-1β, IL-2, TNF-α, IFN-ϒ, MCP-1, MIP-1α, M-CSF, VEGF cytokines and expression of

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MMP-9 gene. The surface chemistry of the CNF scaffolds elicited a modest effect on cytokine

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production and did not shift the inflammatory profile in vitro or in vivo. The decisive role in development of the foreign body reaction was the slow degradation of the CNF scaffolds.

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ACCEPTED MANUSCRIPT 1. Introduction The conventional use of biomaterials as inert implantable devices has shifted to more complicated applications such as drug delivery and tissue engineering [1]. As a result, the biocompatibility paradigm of biomaterials has also shifted, from the absence of adverse tissue reactions towards the

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functional performance of the implanted biomaterials, with an appropriate host response in a

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specific application [1, 2]. In tissue engineering, the classical nontoxic biomaterials are engineered

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to provide 3D porous scaffolds that mimic the extracellular matrix, guiding cell attachment, proliferation and differentiation [1, 3]. Engineering these conventional materials to act as scaffolds

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requires physicochemical modifications, including surface chemistry, size, shape and porosity, all

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of which can affect the host response [4, 5]. Moreover, for successful tissue regeneration, scaffolds with tunable degradation are essential [6]. However, degradation of the scaffolds further adds to

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the complexity of the host response, as the degradation products can in turn provoke the

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inflammatory response [7]. Generally, the host response is initiated and maintained by the production of cytokines released from activated immune cells [8, 9]. These inflammatory cytokines

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are usually categorized as either pro- or anti-inflammatory: their interactions dictate the healing

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process by recruiting, differentiating and polarizing immune cells [8, 10]. For new medical applications therefore, investigating the host response to new forms of

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conventional materials is crucial. In this context, nanocellulose can be an interesting example. Cellulose is the most abundant polysaccharide biopolymer on earth and exists in plants, algae and some marine animals and is produced by certain bacteria [11]. Cellulose and its derivatives, such as regenerated cellulose sponges, are commercially available and commonly used in various biomedical applications [12, 13]. These cellulosic materials are usually prepared by extensive harsh processes which destroy the nanoscale structures of the cellulose [14]. As fabrication techniques have progressed, individualized nanoscale cellulose fibers have been successfully isolated and 3

ACCEPTED MANUSCRIPT cellulose has been reintroduced into the biomedical field in new nanoscale forms [15]. The most commonly used nanocellulose material in tissue engineering is bacterial nanocellulose (BNC) [16]. Recently, wood-based cellulose nanofibrils (CNF) hydrogels have been shown to have great potential for 3D culture and bioprinting of stem cells [17, 18]. Compared with conventional

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celluloses, these nanocelluloses exhibit improved characteristics including high specific surface

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area, rheological properties, surface chemical reactivity and good mechanical reinforcement [19].

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With respect to biocompatibility, cellulose derivatives are reported to evoke mild to moderate foreign body reactions (FBR) in vivo [20-22]. The host response to BNC has been investigated and

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found to be acceptable for various applications [16, 23, 24]. However, there is little or no

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information about the long-term biocompatibility of wood-based CNF scaffolds in vivo. There are few scientific reports of the in vitro and in vivo inflammatory responses of cellulose materials in

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general and of CNF in particular. The lack of detailed knowledge of CNF–immune system

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interactions may become a major barrier to developing effective cellulose-based applications in tissue engineering.

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The aim of the current study was to investigate the inflammatory response of porous scaffolds made

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of our recently developed CNF hydrogels, in which TEMPO-oxidation and carboxymethylation were used to prepare CNF materials with varying surface chemistries [25]. Immortal human

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monocyte/macrophage U937 cells were used to profile the inflammatory cytokines in vitro, while the early and late host responses to the scaffolds were investigated in rats after 4 and 30 days utilizing PCR and Bioplex bead arrays. Finally, the foreign body reaction was assessed histologically for up to 6 months. To the best of our knowledge, this is the first in vitro and in vivo report profiling the inflammatory cytokines produced in response to wood-based nanocellulose scaffolds.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1.

Scaffold preparation and characterization

TEMPO-oxidized (TO-CNF) and carboxymythelated (CM-CNF) hydrogels were prepared as described previously [25]. Briefly, TEMPO-oxidized CNF was prepared

using a 2,2,6,6-

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tetramethylpiperidinyl-1-oxyl (TEMPO), sodium bromide and sodium hypochlorite system

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(Sigma-Aldrich, St Louis, MO, US) to oxidize a bleached softwood kraft pulp (A mixture of Pine

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(25%) and Spruce (75%) from Södra Cell, Växjö, Sweden) [26]. For carboxymethylation, the pulp was impregnated in a solution of monochloroacetic acid and isopropanol (Sigma-Aldrich). The

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carboxyl groups were converted to the sodium form by soaking the pulp in a 4% NaHCO3 solution

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and the pulp was finally filtered and washed with deionized water [27]. After both chemical treatments, the pulp fibers were homogenized at 1000 bar pressure with a Rannie homogenizer

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(APV, SPX Flow Technology, Silkeborg, Denmark).

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Electric conductivity titration was used to determine the content of carboxyl and aldehyde groups introduced to the surface of the CNF [26]. After chemical treatment, dry fibers were washed with

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NaCl solution with the pH adjusted to 2.5. The titration was then performed with NaOH solution

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at a rate of 0.1 ml/min until the pH reached approximately 11. The conductivity of the samples was measured using a Metrohm 856 Conductivity Module. The carboxylate content was calculated

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from the titration curve. To calculate the aldehyde content, the same analysis was done after oxidation of aldehyde groups to carboxyl groups with NaClO2. The aldehyde content is the difference in carboxylate content before and after oxidation [25]. To evaluate the fibrillar structure of the CNF materials, thin films were prepared by drying CNF dispersion on coverslips at room temperature. Films were then analyzed by an atomic force microscope (diMultiMode V AFM, Bruker, U.S.A.).

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ACCEPTED MANUSCRIPT Finally, to prepare the 3D porous scaffolds, the prepared hydrogels were cast into suitable well plates and frozen at -20°C for 24 h and then freeze-dried for 24 h. The structure of the scaffolds was characterized by scanning electron microscopy (SEM, JEOL JSM-7400F, Japan). Microcomputed tomography (µ-CT, Skyscan 1172VR, Kontich, Belgium) was used to evaluate the

Water uptake and scaffold degradation

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2.2.

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porosity of the prepared scaffolds as described previously [28].

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To evaluate the water uptake, the dried scaffolds were weighed (Wd = weight of dry samples) and then immersed in 10 ml phosphate buffered saline (PBS; Life Technologies, Carlsbad, CA, USA)

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at 37°C for 72 h [10]. At each time point, the scaffolds were weighed (Ws = weight of swollen

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samples) after wiping off the excess surface water. The water uptake was calculated as follows:

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Water uptake (%)=

(Ws - Wd) 𝑥 100 Wd

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The in vitro degradation of the scaffolds was determined in 20 ml PBS at 37°C under shaking conditions (60 rpm). Dried scaffolds were weighed (W0) and incubated in PBS for 90 days. The

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PBS was replaced twice a week. After 90 days, the samples were freeze-dried and weighed (W90).

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The degradation was calculated as the change in the mass using the following equation;

Mass loss (%)=

(W0- W90) 𝑥 100 W0

Cell culture

For evaluation of the inflammatory responses to the prepared CNF scaffolds, cells of the immortalized monocyte U937 cell line (ATCC; Manassas, VA, USA) were used. The cells were cultured in DMEM growth medium (Gibco, Thermo Fisher Scientific) supplemented with 10 % FBS (HyClone, GE healthcare, Utah, USA) and 1% antibiotics (100 U/ml penicillin and 0.1 mg/ml

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ACCEPTED MANUSCRIPT streptomycin, HyClone, GE healthcare) at 37°C in 5% CO2 [29]. U937 cells between passages 8 and 18 were used for differentiation into macrophage-like cells, as recommended by the supplier. The differentiation was done using 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-

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Cell viability, proliferation and morphology on scaffolds

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Aldrich) (Supplementary Information) [30].

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U937 cells were seeded onto the various CNF scaffolds (1 × 105 cells/ml) in the presence of the

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PMA to initiate and maintain the differentiation of the cells. For cell viability, proliferation and

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attachment, scaffolds (10 mm × 3 mm) were used in 24-well plates. Empty wells of tissue culture plates (TCP; Nunc, Thermo Fisher Scientific) served as controls [31]. Cell viability after 2 days

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was analyzed by live/dead staining (Invitrogen, Carlsbad, CA, USA). Seeded scaffolds were

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washed with PBS and incubated in a working solution containing EthD-1 and Calcein-AM for 30 min at room temperature and then imaged with a fluorescence microscope (Nikon, Eclipse, 80i,

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Tokyo, Japan). The relative proliferation was evaluated by DNA assay (Quant-iTTM PicoGreen®,

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Invitrogen). At days 1 and 3, the scaffolds were washed with PBS, covered with 0.1% TritonX/PBS and then stored at −80°C. After two freezing-thawing cycles and sonication for 60 seconds,

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50 µL of each sample was transferred to 96-well plates. Finally, 150 µL PicoGreen® working

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solution was added to each well and the fluorescence at 480/520 nm was measured with a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Germany). To study cell morphology, seeded scaffolds at day 2 were washed with PBS and fixed with 4% paraformaldehyde. PhalloidinAtto488 (Sigma-Aldrich) in PBS (dilution 1:50) was added to the wells for 40 min and then examined using a fluorescence microscope. For SEM analysis, scaffolds were fixed with 3% glutaraldehyde, vacuum dried, sputter-coated with platinum and imaged at 5 kV.

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ACCEPTED MANUSCRIPT 2.5.

In vitro cytokine measurement

For detection of the inflammatory cytokines, scaffolds (20 mm × 4 mm) were seeded with U937 cells (5 × 105) in 6-well plates (5 ml medium). Cells on tissue culture plates (TCP; Nunc, Thermo Fisher Scientific) were used as controls. The inflammatory cytokines in the supernatant were

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quantified after 1 and 3 days, with a Bio-Plex Human 27-plex kit (Bio-Rad Inc., Hercules, CA,

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USA), using a Multiplex System (Bio-Plex®200, Bio-Rad) according to the manufacturer's

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instructions [10]. The full names and functions of these cytokines are listed in Supplementary Table 1. Based on the findings of several reports [8, 10, 31], certain cytokines were selected and grouped

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into 3 categories according to their roles in foreign body reactions: 1) Pro-inflammatory cytokines

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including IL-1β, IL-2, IL-6, IL-8, IL-12, IFN-ϒ, TNF-α and RANTES; 2) Anti-inflammatory cytokines including IL-1Ra, IL-4, IL-10 and IL-13 and 3) Chemokines and growth factors

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including MCP-1, MIP-1α, MIP-1β, CXCL-1, GM-CSF, M-CSF, PDGF, FGF and VEGF. Subcutaneous implantation in rats

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2.6.

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All the animal experiments were conducted in accordance with the guidelines of the European Convention for the Protection of Vertebrates used for Scientific Purposes. The study protocol was

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approved by the Norwegian Animal Research Authority (FOTS Reference no: 8035). To serve as

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a biocompatible and degradable control, gelatin scaffolds crosslinked with genipin were prepared and used (Supplementary Information). Scaffolds (10 mm × 4 mm: 3 scaffolds/animal) were subcutaneously implanted in the dorsal area of 28 female Wistar rats (weight: 200 g, age 16 weeks) as previously described [32]. At predetermined time points, the samples were explanted and stored in RNAlater (Invitrogen) at -80°C for further investigations.

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ACCEPTED MANUSCRIPT 2.7.

In vivo gene analysis by a customized PCR array

Total RNA was isolated from the in vivo samples after 4 and 30 days using a TRIzolV® reagent (Gibco BRL, Carlsbad, CA, USA) as described previously [33]. The reverse transcription was done according to the manufacturer’s instructions using a commercially available kit (Rt2 First Strand

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Kit, Qiagen Alameda, CA, USA). Rt2 SYBR Green/Rox qPCR Mastermix and a customized Rt2

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Profiler PCR Array (Qiagen) including selected inflammatory genes were used. The PCR was

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performed utilizing a StepOne Plus real time PCR system (Applied Biosystems, Carlsbad, CA, USA) as reported before [32]. GAPDH served as the endogenous reference and gelatin scaffold at

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day 4 served as the reference control sample when analyzing the gene expressions. The full names

In vivo cytokine measurement

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and functions of these genes are listed in Supplementary Table 1.

The explanted scaffolds were sonicated (Bandelin Sonopuls, Berlin, Germany) in RIPA buffer

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containing protease inhibitor cocktail (Thermo Fisher Scientific). Samples were then centrifuged

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at 14000 × g for 10 min and the supernatants were transferred to fresh tubes. The total protein concentration in supernatants was determined using a BCA Protein Assay Kit (Thermo Fisher

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Scientific), following the manufacturer’s instructions to standardize the concentrations. Finally,

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Bio-Plex Rat 24-plex kit (Bio-Rad) was used with a Multiplex System (Bio-Plex®200, Bio-Rad) according to the manufacturer's instructions [34]. The cytokines were also categorized as pro-, antiinflammatory or chemokines/ growth factors as mentioned earlier. 2.9.

Histology

The scaffolds were harvested with the surrounding tissue at 4, 30 and 180 days and fixed with 4% paraformaldehyde (Merck, Germany). Samples were then paraffin embedded, serially sectioned (3-5 µm) and stained using hematoxylin and eosin (H&E) as previously reported [28]. 9

ACCEPTED MANUSCRIPT 2.10. Statistical Analysis Statistical analysis was undertaken using SPSS software (IBM, Armonk, NY, USA). The tests applied were One-way ANOVA with a Tukey's post-hoc comparison of the mean. Data are expressed as the mean ± standard deviation (SD). Differences were considered statistically

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significant at p ≤ 0.05.

Scaffold fabrication and characterization

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3.1.

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3. Results

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The prepared TO-CNF and CM-CNF samples had solid content of 1.06 ± 0.01 and 1.07 ± 0.01% respectively. This low solid content was enough to form a weak gel-like material as shown in Fig

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1. Structural investigations by AFM revealed that the prepared materials have a nanoscale fibrillar

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network (Fig. 1). The electric conductivity titration demonstrated that the TO-CNF had aldehyde and carboxyl groups (211±60 and 764±60 µmol/g respectively). The CM-CNF had carboxymethyl

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(384 764±26 µmol/g) and less carboxyl groups (58±1 µmol/g). After freeze-drying, the porosity of

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the prepared CNF scaffolds was evaluated descriptively by SEM and quantitatively by µ-CT (Fig 2). To create ice crystals to act as porogens, the prepared hydrogels were frozen at -20°C prior to

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freeze-drying. The resultant scaffolds exhibited internal structures with interconnected pores of

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various diameters, ranging from 7 µm to more than 590 µm.

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Figure 1. Macroscopic images of the prepared gel-like CNF materials and their nanoscale fibrils

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by AFM.

Figure 2. Structural analysis by SEM and pore size distribution by µCT of prepared CNF scaffolds by freeze-drying. Both scaffolds had pore size between 7 and 590 µm (n = 5). 11

ACCEPTED MANUSCRIPT 3.2.

Water uptake and in vitro degradation

The water uptake was measured as mass gain over 72 h as shown in Fig. 3. Both CNF scaffolds demonstrated high water uptake with averages of 2520 ±307 % for TO-CNF and 2496 ±328 % for CM-CNF. The in vitro degradation of the samples was also determined as a function of mass

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change over 90 days. The structural characterization by SEM and µ-CT disclosed collapsing

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porosity and changing volume of the samples after 90 days. Before soaking in PBS, the total

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porosity percentages of TO-CNF and CM-CNF were 93.4 ± 1.6 and 93 ± 1.5% respectively. After 90 days in PBS, the porosity decreased to 81.8 ±1.1 % for TO-CNF and 83.7 ± 5.3 % for CM-CNF,

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indicating the collapse of the structure. However, both scaffolds showed a very small percentage

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of mass loss over the period of 90 days: 6.7% ±1.2 for TO-CNF and 6.5 ±3.6 for CM-CNF.

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ACCEPTED MANUSCRIPT Figure 3. In vitro degradation and water uptake of CNF scaffolds. (A) SEM micrographs and (B) µCT images showing the collapsed structure of CNF scaffolds after 90 days in PBS. (C) Changes in the total porosity by µCT after immersing in PBS for 90 days (n= 5). (D) Degradation by mass loss after 90 days in PBS (n = 5). (E) Water uptake capacity of CNF scaffolds during 72 h in PBS

Cell-scaffold interactions

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3.3.

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(n = 5).

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Phase contrast microscope images and phalloidin stained cells indicated that PMA-treated cells

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underwent morphological changes (Fig. 4 and Supplementary Fig. 1). Unlike the cells on the CMCNF scaffolds, those on TO-CNF scaffolds exhibited larger and more irregular shapes, with

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extended filopodia and ruffled surfaces. Although there was no increase in proliferation from day

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1 to day 3, the live/dead assays confirmed that cell viability was maintained. There were no

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rates or viability of the cells.

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significant differences between TO-CNF and CM-CNF scaffolds with respect to the proliferation

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ACCEPTED MANUSCRIPT Figure 4. In vitro PMA-treated U937 cell-scaffold interactions. (A) Cell viability by live/dead stain after 1 and 3 days. Living cells stained green and dead cells stained red. (B) Proliferation of cells cultured on CNF scaffolds for 3 days, relative to cells cultured on TCP (n =4). (C-D) Morphological changes by phalloidin staining and SEM analysis. Cells cultured on TO-CNF showed extended

In vitro cytokine measurement

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filopodia while cells cultured on CM-CNF had smooth surface.

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Twenty-seven inflammatory cytokines were measured in the supernatant of the U937 cells cultured

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on CNF scaffolds (Fig. 5). The concentrations of these cytokines were presented in color map format to visualize the production patterns over time (Fig. 5A). As individual cytokine production

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can be difficult to interpret, selected cytokines were categorized into pro-inflammatory,

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chemokines/growth factors and anti-inflammatory cytokines (Supplementary Fig. 2). At day 1, proinflammatory cytokine production was significantly less from the cells cultured on TCP than on

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CM-CNF scaffolds. Similarly, production of chemokines/growth factors in the TCP group at day

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1 was significantly less than both CNF scaffolds; production of anti-inflammatory cytokines from all groups was comparable, with no significant differences. In contrast to the trend at day 1, the

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TCP promoted more production of all integrated cytokines at day 3. With respect to production of

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chemokines/growth factors and anti-inflammatory cytokines by TCP and both CNF scaffolds at day 3, the differences were significant. In addition, there was no significant difference between TO-CNF and CM-CNF at either 1 or 3 days. Individually, the production of most of the cytokines increased from day 1 to day 3 in all groups, with the highest production levels for IL-8 (Fig. 5B). IL-13 was not detected after day 1 in any of the groups and was then found at low concentration after 3 days, with no significant intergroup differences. After 3 days, IL-1Ra, MCP-1, MIP-1α, MIP-1β, RANTES and VEGF were all present at concentrations above 700 pg/ml.

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ACCEPTED MANUSCRIPT Statistically, there were 8 instances at day 1 and 11 instances at day 3 of significant differences between TCP and CNF groups. The only significant difference between TO-CNF and CM-CNF was detected for the production of RANTES on day 3, where CM-CNF induced higher concentrations than TO-CNF. Unlike IL-10 and MCP-1, the concentrations of IL-1β, IFN-ϒ, TNF-

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α, MIP-1α, MIP-1β and IL-4 were significantly lower in TCP than in the CNF groups at day 1. On

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the other hand, at day 3, IL-6, IL-12, IFN-ϒ, TNF-α, MIP-1β, VEGF, IL-Ra, IL-4 and IL-10 were

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significantly higher in TCP than in the CNF groups. In summary, the cells cultured on CNF groups, regardless of their chemistry, produced less pro-inflammatory and chemokines/growth factors than

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cells cultured on the TCP control group after 3 days.

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ACCEPTED MANUSCRIPT Figure 5. In vitro profiling of cytokine production in supernatant of PMA-treated U937 cells cultured on CNF scaffolds after 1 and 3 days. Tissue culture plates (TCP) served as control. (A) Heat map presentation of produced cytokines over time. (B) Integrated cytokines after grouping into pro-inflammatory, chemokine/growth factors and anti-inflammatory cytokines based on their

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role in the foreign body reaction. (n =4. ∗p ≤ 0.05 ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). In vivo cytokine gene expression

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The expression of the genes was presented in color map format as shown in Fig. 6A. Relative to

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the gelatin scaffold at day 4, the differences in expression among 18 genes were significant only for IL-Ra, for which expression by both TO-CNF and CM-CNF scaffolds was higher than for the

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gelatin scaffold (Fig. 6B). Furthermore, 4 pro-inflammatory genes (IL-2, IL-6, TNF-α, RNATES),

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2 chemokines/growth factor genes (MIP-α, VEGF) and MMP-9 gene were slightly upregulated by TO-CNF. As well as these genes, CM-CNF slightly upregulated 4 extra genes: IFNa1, MCP-1,

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FGF and the anti-inflammatory IL-13 after 4 days. At day 30, CNF scaffolds exhibited general

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downregulation of most of the genes. On the other hand, at day 30 the gelatin scaffold showed upregulation of 10 genes, including IL-2, IFNa1, TNF-α, RNATES, MIP-α, VEGF, FGF2, MMP-

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9, IL-1Ra and IL-10. However, the only significant intergroup differences were for IL-1Ra and

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MMP-9, for which both CNF scaffolds had the highest expression at day 30.

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Figure 6. In vivo cytokine gene expression in response to implanted scaffolds after 4 and 30 days

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post-implantation. (A) Heat map presentation of produced cytokines over time. (B) Integrated cytokines after grouping into pro-inflammatory, chemokine/growth factors and anti-inflammatory

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cytokines base on their role in the foreign body reaction. Expression is presented as 2

−ΔΔCt

after

In vivo cytokine measurement

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3.6.

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normalization to GAPDH of the gelatin scaffold at day 4. (n =4. ∗p ≤ 0.05 ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).

Similar to the in vitro profiling, the concentrations of the 24 cytokines are presented in color map format (Fig. 7A). At day 4 and after categorization, the gelatin scaffold stimulated significantly higher production of the pro-inflammatory cytokines than CNF scaffolds, while TO-CNF promoted the highest production of chemokines/growth factors (Supplementary Fig. 2). At day 4 there were no significant intergroup differences in the production of anti-inflammatory cytokines. After 30 days, there was an overall reduction in production of the pro-inflammatory and chemokines/growth

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ACCEPTED MANUSCRIPT factors in the gelatin and TO-CNF groups while the CM-CNF scaffold exhibited almost a steady production profile. Unlike gelatin, both CNF scaffolds promoted significantly higher production of the anti-inflammatory cytokines, with no differences between TO-CNF and CM-CNF at day 30. Individually, at day 4, the production of IL-1β was the highest from all groups with concentrations

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above 5000 pg/ml (Fig. 7B). The concentrations of MCP-1 and the anti-inflammatory IL-10 in all

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groups, IL-6 and MIP-1α in the gelatin scaffold, and VEGF in the gelatin and the TO-CNF scaffolds

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displayed concentrations above 2500 pg/ml. After 30 days, IL-1β in all groups, IFN-ϒ in the gelatin

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and CM-CNF, MCP-1 and IL-10 in TO-CNF and CM-CNF and VEGF in the TO-CNF were present above 1000 pg/ml. On the other hand and in contrast to the in vitro results, no production of G-

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CSF was detected in any of the groups at 4 and 30 days. Moreover, GM-CSF and IL-13 were not

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detected in any of the groups at day 30.

Statistically, there were 6 instances at day 4 where the differences between gelatin and one or both

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CNF groups were significant. Unlike VEGF, the concentrations of the pro-inflammatory IL-1β, IL-

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6, IL-12, MIP-1α and CXCL-1 and M-CSF were significantly higher in the gelatin than in the CNF

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groups at day 4. At day 30, the concentrations of the TNF-α, MIP-1α, M-CSF, VEGF, and IL-10 cytokines were significantly less in the gelatin scaffold. Except for IFN-ϒ, the gelatin scaffold

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exhibited either decreased or undetected production of all cytokines at day 30. Significant differences between TO-CNF and CM-CNF were detected for the production of MCP-1, CXCL-1, VEGF, where CM-CNF induced less production than TO-CNF.

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Figure 7. In vivo profiling of cytokine production in response to implanted scaffolds after 4 and

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30 days in rats. (A) Heat map presentation of the produced cytokines over time. (B) Integrated

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cytokines after grouping into pro-inflammatory, chemokine/growth factors and anti-inflammatory

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cytokines base on their role in the foreign body reaction. (n =4. ∗p ≤ 0.05 ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). Host tissue reactions

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The in vivo degradation of the porous gelatin, TO-CNF and CM-CNF scaffolds was evaluated over a period of 180 days of subcutaneous implantation in rats (Fig. 8). After 30 days, the gelatin scaffold almost disappeared, while both CNF scaffolds showed slow degradation with a slight decrease in cross-sectional area and a more apparent decrease in height. However, after 180 days, both CNF scaffolds maintained their round shape while the gelatin scaffold disappeared completely. CNF scaffolds stimulated blood vessel formation particularly at day 30.

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Figure 8. Fate of scaffolds after 4, 30 and 180 days. A gelatin scaffold crosslinked with genipin

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was used as a control. The gelatin scaffold showed fast degradation while both CNF scaffolds

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maintained their rounded structure up to 180 days. CNF scaffolds stimulated blood vessel

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formation particularly at day 30. Representative sections (H&E-staining) of the early host response (day 4 time point) of the different scaffolds are presented in Fig. 9. All the scaffolds showed cell infiltration into the pores; however, more cells were able to invade the walls of the pores of the gelatin scaffold. The cells colonizing the scaffolds at day 4 were mostly neutrophils and monocytes. Furthermore, a thin fibrous encapsulation was detected around all the scaffolds.

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Figure 9. Representative H&E stained sections of early host reaction in response to scaffolds at

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day 4 after implantation. All scaffolds developed a thin fibrous capsule. The outer region of all

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scaffolds was invaded with immune cells including lymphocytes, monocytes and neutrophils. Unlike CNF scaffolds, pore wall of gelatin scaffold was broken by the cells indicating early degradation of gelatin.

The long-term host responses (30 and 180-day time points) are presented in Figs. 10 and 11. The residual gelatin was colonized with macrophages and blood vessels without foreign body giant cells (FBGCs). The presence of macrophages and FBGCs was observed in both CNF scaffolds. Unlike the rapid degradation of gelatin, CNF scaffolds retained much of their original shape over 21

ACCEPTED MANUSCRIPT time with some weak signs of degradation such as fibrillation of the walls and small cellulose fragments. Moreover, the pores of the CNF scaffolds were filled, partially at day 30 and entirely at day 180, with granulation tissue composed of collagen matrix, fibroblasts, blood vessels, macrophages and FBGCs. Moreover, a steady vascular fibrous capsule was found around both CNF

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scaffolds at 180 days.

Figure 10. Representative H&E stained sections of late host reaction in response to implanted scaffolds at day 30 after implantation. Gelatin scaffold showed fast degradation as most of the scaffold area was replaced by tissue. Macrophages and blood vessels (BV) colonized the residuals 22

ACCEPTED MANUSCRIPT of the gelatin scaffold. CNF scaffolds stimulated the formation of foreign body reaction. Both CNF scaffolds were surrounded by well-defined fibrous capsule and their pores were partially filled with granulation tissue that contained fibroblasts, macrophages, foreign body giant cells (FBGC),

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blood vessels and collagen matrix.

Figure 11. Representative H&E stained sections of late host reaction in response to implanted CNF scaffolds at day 180 after implantation. CNF scaffolds maintained their shape with some signs of degradation (D) such as delamination of the fibers and fragmentation. Both CNF scaffolds were surrounded by a steady fibrous capsule and their pores were fully filled with granulation tissue with macrophages and foreign body giant cells.

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ACCEPTED MANUSCRIPT 4. Discussion Although the in vivo host response is characterized by a diversity of cell types, the predominant cells at the surface of the implanted scaffolds are the monocyte-derived macrophages [35, 36]. These macrophages orchestrate the immune response by production of various cytokines which

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initiate and maintain the recruitment and activation of the surrounding cells [31, 35, 37]. The U937

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macrophage-like cell line was selected for the study because the primary monocyte-derived

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macrophage cells are reported to have more donor variability and require more time for differentiation [38]. Although the CM-CNF scaffold supported the viability and proliferation of the

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U937 cells, the carboxymethylation was reported to affect cell morphology [25]. Earlier, Hua et al.

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reported the effect of carboxymethylation on macrophage morphology and stated anionic CNF films with carboxymethyl groups stimulated the secretion of TNF-α compared to cationic films

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after 1 day [39]. Moreover, they showed that none of these CNF materials triggered the cells to

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produce IL-10. In contrast to these findings, the present results demonstrated that both CNF scaffolds supported production of anti-inflammatory cytokines comparable to the TCP after day 1

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and increased significantly on day 3. Most importantly, at day 3 both CNF scaffolds stimulated less

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production of pro-inflammatory cytokines and chemokines/growth factors than TCP, which is acknowledged as an appropriate control for monitoring cytokine production from macrophages

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[31]. IFN-ϒ is regulated by the secretion of IL-12 and is known to activate macrophages and regulate IL-1β production [40]. The high production of the neutrophils chemoattractant IL-8 and pro-inflammatory IL-6 in all the groups is likely to be due to the secretion of IL-1β and IFN-ϒ [40, 41]. However, the lack of detection of IL-6 in the TCP group at day 1 may be the result of incomplete differentiation of the U937 cells. It is known that monocytes, if not differentiated to macrophages, secrete IL-8 but not IL-6 [41]. In general, this can explain the obvious increase in the production of all the cytokines from day 1 to day 3 due to the increase in the number of 24

ACCEPTED MANUSCRIPT differentiated cells. With respect to the surface chemistry, the different surface groups on CNF scaffolds resulted in insignificant variations in the production of all cytokines except for RANTES at day 3. In agreement with this finding, Schutte et al. reported that cytokine production from human macrophages was modestly affected by the surface chemistry of nontoxic biomaterials [8].

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Taken together, these findings indicate that the CNF scaffolds trigger a mild inflammatory response

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after 3 days of culture with U937 cells in vitro.

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However, in vitro conditions cannot fully simulate the complex in vivo environment. Hence, the

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early and late inflammatory responses to the CNF scaffolds were investigated in rats, in an attempt to correlate the histological findings with the cytokine production. Freeze-dried porous gelatin

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scaffolds were selected as controls because of the reported rapid degradation and less pronounced

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foreign body reaction [10]. In general, in vivo implantation of scaffolds initiates dynamic cytokinemediated events, including acute inflammation, chronic inflammation, and foreign body reaction

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(FBR) within the first 2 weeks [10, 42]. The acute inflammation in the present study was

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predominated by lymphocytes, monocytes and neutrophils, as shown in the histological sections at day 4. The neutrophils were recruited by the release of IL-8, CXCL-1 and VEGF, as confirmed

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from the gene expression and cytokine production in vitro and in vivo [5, 40]. Moreover, the

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continuous production of RANTES, MCP-1 and MIP chemokines in vitro and in vivo suggests the constant recruitment of macrophages in vivo [8, 40]. Catalán et al. detected neutrophils and macrophages in the lungs of mice as an early inflammatory response to different TEMPO-oxidized CNF concentrations administrated by a single pharyngeal aspiration. They also found a dosedependent increase in the gene expression of TNF-α, IL-1β, IL-6 and RANTES 24 h postadministration [43]. In a similar study, extensive recruitment of neutrophils in the lungs of mice was reported as an acute inflammatory response to exposure to CNC materials derived from wood.

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ACCEPTED MANUSCRIPT Compared to the control mice, there was a significant increase in the production of IL-1α, IL-1β, IL-6, G-CSF, MCP-1, MIP-1α, MIP-1β, and TNF-α [44]. Most importantly, the insignificant difference between the CNF and gelatin scaffolds with respect to the expression of most of the inflammatory genes confirmed an early mild inflammatory response. Significantly, CNF scaffolds

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exhibited higher expression of IL-1Ra, which is known to suppress the pro-inflammatory signals

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[31, 45]. Moreover, both CNF scaffolds, regardless of their surface chemistries, stimulated

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significantly less pro-inflammatory cytokines than the gelatin at day 4, due to less production of IL-1β, IL-6, MIP-1α, CXCL-1 indicating the early mild response of the CNF scaffolds. However,

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when the inflammatory response of the categorized cytokines was compared, TO-CNF produced

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higher concentrations of chemokines/growth factors than CM-CNF on day 4. This may suggest

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that TO-CNF induces more early pro-inflammatory signals than CM-CNF. The late response to implanted scaffolds is characterized by the presence of the foreign body

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reaction (FBR) which includes macrophages, foreign body giant cells (FBGCs) and vascularized

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connective tissue [2, 22, 40]. In general, both degradable and non-degradable materials develop a certain degree of foreign body reaction [20, 36]. However, the reaction disappears as degradation

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proceeds and continues with non-degradable materials, with macrophages orchestrating the

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reaction [40, 46]. The slow degradation of the cellulose is due to the absence of cellulose-degrading enzymes in animals [47]. Moreover, the packed molecular structure of the cellulose retards the breakdown of the cellulose by hydrolysis, as observed from the present in vitro results and in agreement with the results of other studies [48]. At the cellular level, macrophages are known to phagocytose biomaterials, including CNF [35, 36, 43]. The steady presence of activated macrophages is regarded as a late response to slowly degradable scaffolds. Indeed, it was reported that the lack of macrophages resulted in the absence of the typical histological features of the FBR

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ACCEPTED MANUSCRIPT [35]. Importantly, many studies have reported that the macrophage polarization is responsible for coordinating the FBR [36, 46, 49]. M1-polarized macrophages are considered a pro-inflammatory phenotype while M2 is considered an anti-inflammatory phenotype [46, 49, 50]. M1 cells are characterized by the production of IL-1β, IL-2, IL-6, IL-12, MMPs, TNF-α and IFN-ϒ cytokines:

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these were found in higher concentrations in CNF groups than in gelatin at day 30 [51, 52]. On the

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other hand, M2 macrophages are known to release high amount of the anti-inflammatory cytokines

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especially IL-10 [37, 53]. Both CNF supported anti-inflammatory cytokine production comparable to gelatin at day 4 and significantly higher at day 30. Based on the cytokine profile, we may

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inflammatory profile both in vitro and in vivo.

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speculate that CNF scaffolds stimulated both macrophage phenotypes with M1 dominating the

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Histological analysis disclosed a thin fibrous capsule and the absence of FBGCs as early as day 4 in all scaffolds. After 30 days, CNF scaffolds, unlike gelatin, stimulated the formation of FBGCs.

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In addition to the degradation factor, the absence or presence of the FBGCs might be linked to the

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significant differences in the production of IL-6 that attracts macrophages and upregulates MMP inhibitors [9]. Moreover, it is hypothesized that macrophage fusion is an escape mechanism

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regulated by TNF-α to avoid apoptosis [54]. Therefore, the low production of TNF-α from gelatin

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compared to CNF scaffolds can be correlated with the absence of the FBGCs in the remaining gelatin at day 30. IL-4 and IL-13 are also known to regulate macrophage fusion in vitro and in vivo [54, 55]. Furthermore, MacLauchlan et al. detected high levels of MMP-9 during macrophage fusion in vitro and in vivo, which is in accordance with the high gene expression of MMP-9 in CNF scaffolds at day 30 [56]. Moreover, the continuous production of M-CSF and VEGF from the CNF scaffolds can also be linked to the formation of FBR and FBGCs [46]. Dondossola et al. identified a link between FBGCs and VEGF production as a central pathogenic axis driving the host response

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ACCEPTED MANUSCRIPT towards FBR [37]. Interestingly, both CNF scaffolds stimulated the sprouting of new blood vessels, which can be linked to the high in vitro and in vivo secretion of VEGF, FGF and PDGF. As with the in vitro results, the overall reduction of the pro-inflammatory cytokines and chemokines/growth factors in the CNF scaffolds can be attributed to the high production of IL-10 [57]. By stimulating

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the production of IL-10, IL-4, IL-13 signals, CNF scaffolds, if degraded faster, might potentially

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direct macrophages toward an anti-inflammatory phenotype by inhibiting their proliferation and

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activation [51].

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In accordance with the histological findings of the present study, several reports on cellulosic materials, including BNC, have shown a similar chronic phase of inflammation with macrophages,

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FBGCs and fibrous encapsulation [22, 58, 59]. In contrast, however, some reports have shown that

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BNC membranes induce mild inflammatory responses without foreign body reaction [23, 60]. These contradictory findings may be attributable to the different animal species used in the studies,

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as well as the shape and size of the implanted materials [4]. For example, several studies have

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suggested a relationship between scaffold porosity and macrophage polarization [49, 50]. The interconnected porosity of the CNF scaffolds in the current study had bigger pore diameters and

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this could contribute to the development of the later FBR [49].

Additionally, it has been

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hypothesized that the different surface chemistry of biomaterials dictates the host response through the adsorbed proteins on their surfaces [61, 62]. In contrast, it has also been proposed that with the exception of a known toxic material, the initial phases of the host response are similar, regardless of the characteristics of the implanted scaffolds [8, 31]. The late response towards any nontoxic and non-degradable materials has similar endpoints of fibrous encapsulation and foreign body reaction [8, 40]. In agreement, the present in vitro and in vivo results demonstrated that the differences in gene expression and cytokine production between the CNF scaffolds were modest

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ACCEPTED MANUSCRIPT and did not shift the inflammatory profile. Though CM-CNF had a slightly less inflammatory response at day 4, TO-CNF showed obvious reductions in most of the inflammatory cytokines at day 30. However, both materials had the same endpoint of foreign body reaction. The rapid degradation of the gelatin scaffold was the decisive factor shifting the host response towards

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complete resolution.

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5. Conclusions

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This study presents, for the first time, a comprehensive correlation between cytokine production in

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vitro and in vivo and foreign body reaction in response to wood-based cellulose nanofibrils scaffolds. Both TO-CNF and CM-CNF scaffolds demonstrated early mild responses when

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compared to the control groups (TCP in vitro and gelatin scaffolds in vivo). At day 3 in vitro, the

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interaction with the differentiated U937 cells showed comparable production of the categorized pro-inflammatory cytokines and significantly less production of the chemokines/growth factors

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than TCP. At day 4 in vivo, both CNF scaffolds stimulated significantly less production of the pro-

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inflammatory cytokines than the gelatin scaffold. However, the lack of degradation resulted in the formation of a late and well-developed foreign body reaction that was regulated by the secretion of

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different cytokines. In a few instances, there were significant differences between TO-CNF and

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CM-CNF scaffolds with respect to the production of some cytokines. However, both materials had the same endpoint of the foreign body reaction. Clearly, both CNF scaffolds had no toxicity in vitro or in vivo with good capacity to induce repair, which might be useful in cases of impaired wound healing. However, in order to achieve a shift from reparative to more regenerative function, modifications of these CNF materials should be considered, in order to inhibit the FBR and to improve degradation.

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ACCEPTED MANUSCRIPT Acknowledgements This work has been funded by the Research Council of Norway through the NORCEL project, (Grant no. 228147) and Helse Vest projects 502027 and 912048. We would like to thank Ingebjørg Leirset, Randi Sundfjord, Mohammed Yassin and Alexander Sauter for their technical support.

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Disclosures: The authors declare no conflicts of interest.

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Graphical Abstract

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Highlights Two nanocellulose scaffolds were prepared by TEMPO-oxidation and carboxymethylation. Both scaffolds had no toxicity in vitro or in vivo. Both scaffolds had early mild inflammatory responses.

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Both scaffolds had late similar foreign body reactions due to the slow degradation.

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The different surface chemistries had a modest impact on the host response.

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